Multi-chain chimeric polypeptides and their use in treating liver disease - Patents.com

Abstract

Provided herein are multi-chain chimeric polypeptides and their uses in treating liver disease.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 63 / 232,140, ​​filed August 11, 2021, and U.S. Patent Application No. 63 / 330,757, filed April 13, 2022, the contents of both of which are incorporated herein in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file titled 47039-0025WO1_SL_ST26.XML. The XML file, created on August 9, 2022, is 83,507 bytes in size. The data within the XML file is incorporated herein by reference in its entirety.

[0003] Technical Field The present disclosure relates to the field of biotechnology, and more particularly to antigen binding molecules and the treatment of liver disease. [Background technology]

[0004] background Tissue factor (TF), a 263-amino acid integral membrane glycoprotein with a molecular weight of approximately 46 kDa and the trigger protein of the extrinsic blood coagulation pathway, is the primary initiator of coagulation in vivo. Tissue factor is not normally in contact with circulating blood and initiates the coagulation cascade upon exposure to circulating serine protease factors. Vascular injury exposes subendothelial cells expressing tissue factor, resulting in the formation of a calcium-dependent, high-affinity complex with pre-existing plasma factor VIIa (FVIIa). Binding of the serine protease FVIIa to tissue factor promotes the rapid cleavage of FX to FXa and FIX to FIXa. The resulting FXa and the proteolytic activity of the activated membrane surface then inefficiently convert small amounts of prothrombin to thrombin. Thrombin generated by FXa initiates platelet activation, activating trace amounts of pro-cofactors factor V (FV) and factor VIII (FVIII) to the active cofactors, factor Va (FVa) and factor VIIIa (FVIIIa). FIXa complexes with FVIIIa on the platelet surface to form the intrinsic tenase complex, which leads to the rapid generation of FXa. FXa complexes with FVa to form the prothrombinase complex on the activated platelet surface, which leads to the rapid cleavage of prothrombin to thrombin.

[0005] In addition to the tissue factor-FVIIa complex, recent studies have shown that the tissue factor-FVIIa-FXa complex can activate FVIII, providing additional levels of FVIIIa during the initiation phase. While the extrinsic pathway is most important in initiating coagulation through activation of limited amounts of thrombin, the intrinsic pathway sustains coagulation by dramatically amplifying the initial signal.

[0006] Much of the tissue factor expressed on the cell surface is "encoded" and must be "decoded" to fully participate in coagulation. The mechanism by which cell surface tissue factor is "decoded" is currently unknown, but exposure of anionic phospholipids plays a major role in this process. Healthy cells actively sequester anionic phospholipids, such as phosphatidylserine (PS), in the inner leaflet of the plasma membrane. Upon cell damage, activation, or cytosolic Ca 2+ Following an increase in tissue factor levels, this bilayer asymmetry is lost, resulting in increased PS exposure on the outer leaflet, which increases the specific activity of the cell surface tissue factor-FVIIa complex. Although PS exposure is known to decrease the apparent Km for activation of FIX and FX by the tissue factor-FVIIa complex, additional mechanisms may include conformational rearrangements of tissue factor or tissue factor-FVIIa and subsequent exposure of substrate-binding sites. Summary of the Invention

[0007] overview Provided herein are methods for treating liver disease or metabolic syndrome in a subject, the method comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising: (i) a second domain of the pair of affinity domains, and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through binding of the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII. In some embodiments of any of the methods described herein, the liver disease is selected from the group of fatty liver disease, hepatic steatosis, acute hepatic porphyria, Alagille syndrome, alcohol-related liver disease, alpha-1 antitrypsin deficiency, autoimmune hepatitis, benign liver tumors, cholangiocarcinoma, biliary atresia, Budd-Chiari syndrome, cirrhosis, Crigler-Najjar syndrome, galactosemia, Gilbert syndrome, hemochromatosis, hepatic encephalopathy, hepatitis A, hepatitis B, hepatitis C, hepatorenal syndrome, intrahepatic cholestasis of pregnancy (ICP), lysosomal acid lipase deficiency (LAL-D), liver cysts, liver cancer, neonatal jaundice, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis (PFIC), Reye's syndrome, type 1 glycogen storage disease, and Wilson's disease. In some embodiments of any of the methods described herein, the metabolic syndrome is selected from the group of coronary heart disease, pulmonary disease, gallbladder disease, dyslipidemia, hypertension, type 2 diabetes, dementia, cancer, gynecological disorders including polycystic ovary syndrome, osteoarthritis, pancreatitis, idiopathic intracranial hypertension, stroke, and cataracts.

[0008] A method for reducing one or more of the rate of progression from nonalcoholic fatty liver disease (NAFL) to nonalcoholic steatohepatitis (NASH), from NASH to cirrhosis, and from cirrhosis to hepatocellular carcinoma, comprising administering to a subject identified or diagnosed as having NAFL, NASH, or cirrhosis: (a) a first chimeric polypeptide comprising: (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain of a pair of affinity domains; and (b) a chimeric polypeptide comprising: (i) a second domain of the pair of affinity domains; Also provided herein are methods comprising administering a therapeutically effective amount of a multi-chain chimeric polypeptide comprising (i) a first chimeric polypeptide and (ii) a second chimeric polypeptide comprising a first domain and a second domain of a pair of affinity domains, wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII. In some embodiments of any of the methods described herein, the method results in a reduction in the rate of progression from NAFL to NASH. In some embodiments of any of the methods described herein, the method results in a reduction in the rate of progression from NASH to cirrhosis. In some embodiments of any of the methods described herein, the method results in a reduction in the rate of progression from cirrhosis to hepatocellular carcinoma.

[0009] Also provided herein is a method of reducing inflammation in the liver of a subject, comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising: (i) a second domain of the pair of affinity domains, and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through binding of the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII.

[0010] Also provided herein is a method of reducing hepatic gluconeogenesis in a subject, comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising: (i) a second domain of the pair of affinity domains, and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through binding of the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII.

[0011] Also provided herein is a method of reducing lipogenesis in the liver of a subject, comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising: (i) a second domain of the pair of affinity domains, and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through binding of the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII.

[0012] Also provided herein is a method of reducing hepatocellular senescence in the liver of a subject, comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising: (i) a second domain of the pair of affinity domains, and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through binding of the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII.

[0013] Also provided herein is a method for rebalancing metabolic function in the liver of a subject, comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising: (i) a second domain of the pair of affinity domains, and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through binding of the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII.

[0014] Also provided herein is a method of modulating expression of one or more genes in Tables 1-4 in the liver of a subject, comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising: (i) a second domain of the pair of affinity domains, and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through binding of the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII. In some embodiments of any of the methods described herein, the administration results in a decrease in expression of one or more genes in the liver of the subject selected from the group consisting of ACSS1, RETN, SLC2A4, PDK4, PNPLA3, GADD45B, PPARGC1A, CAV1, ENDOD1, REG3G, IGHG3, IGHG2B, SCGB3A1, GLYCAM1, IGHG2C, IGKC, LTF, MS4A1, JCHAIN, CD19, IGHM, IFI27L2A, ACKR3, LSP1, PMEPA1, CORO1A, GPX3, MYH8, NPPA, TCAP, FLNC, SLC36A2, MYH6, ACTC1, ACTA2, and TPM2, compared to the expression level of the one or more genes in the subject before the administration. In some embodiments of any of the methods described herein, the administration results in an increase in expression of one or more genes in the liver of the subject selected from the group consisting of SLC34A2 and CISH, compared to the expression level of the one or more genes in the subject before the administration. In some embodiments of any of the methods described herein, the administration results in an increase in expression of one or more genes in the liver of the subject selected from the group consisting of CSF3R, IFI27L2A, GM17066, GNL3, FABP1, GM14303, AURKA, RPL14-PS1, QTRT2, G6PC, C8B, DYNLL1, LCN2, LRG1, CEBPD, COL4A3, ST3GAL5, RSAD2, 9330162G02RIK, PINX1,SRA1、SPATA2L、PNRC1、MUP20、IL6RA、APOA1、IL1B、WDR54、CTCFLOS、GM16973、4632427E13RIK、IGHG2B、TGFB1I1、SELENBP2、SEMA6B、NEXN、ZFP653、NOB 1、PCK1、FAM25C、MAPK15、GM16551、ESM1、RPL37RT、FAM133B、PDE8B、TUT1、S 100A11、PDILT、PPARD、IER2、GM15401、MX2、WNK4、G0S2、BC005561、AA986860 、JDP2、GM26982、NOP58、ACTB、GM14586、RPP38、GM13436、NT5DC2、IMPDH1、CYTIP、AI846148、CHKA、GM37963、NR0B2、CYP4A32、ALKBH2、FAU-PS2、PPP1R15 A、KLF2、SLC25A22、GM13341、IGHM、SATB1、SNRPF、DNASE1L2、CD3EAP、GM278 8、DANCR、ZFP612、NOP56、JUND、ID1、HSPB1、KLHDC8A、KLF10、ANGPT2、THBS1、 GM44891、GM9752、ABLIM3、PTGES、GM28438、2410002F23RIK、FOSL2、CRIP3、 JUN、ALAS1、GM2000、RHOC、LMCD1、GM2061、GM42595、GM11478、IKZF2、PNLDC1 、COMTD1、SNORA31、COL20A1、AKAP12、C1QTNF12、1810032O08RIK、2310033P 09RIK、GM47528、SERPINE2、NPFF、SERPINA3K、RFXANK、IGKV5-39、NAB2、MAFF 、CEP85、CSAD、LTB4R1、1810012K08RIK、BCL7C、NRBP2、NLE1、ALKBH1、ARID5 A、CFAP43、GM45767、CD8A、PPRC1、GM26870、TMC7、BCL6B、GM16348、GM26981、 SLC16A3、TNFRSF12A、CYP2J9、NR4A2、MMP9、MIR17HG、TMEM191C、PCDH11X、HILPDA、RAPGEF4、GM17300、SLC25A47、KCNJ2、NYAP1、LAX1、RPS19-PS3、HES1、RGS16, DUSP1, GM43323, ASB4, MUC6, GM15502, UNG, FOXQ1, GM17936, UBE2C, SLC16A6, MIR7052, NLRP12, GM14286, FGF21, KLF5, GM37969, PF4, GM21738, H OTAIRM1, GM6493, LOR, MFSD2B, MATK, SYNE4, GM44694, TRBC1, GM37274, PLN, CXCR4, PHF24, SNORD104, SERPINA7, RGS4, TCIM, EGFR, GM37760, FBXL22, TE DC2, ENHO, GM26917, GM43775, 4833411C07RIK, GM45053, INHBB, OPN3, SNHG15, B230206H07RIK, KCNE3, GM43305, C530043K16RIK, KLF4, LEPR, JCHAIN, T SKU, LGALS4, PCP4L1, GM44829, DUSP8, GM44620, IGFBP1, JUNB, GM32017, GM2814, GM37144, MYADML2OS, GM37666, HDC, SLFN4, A530041M06RIK, GM43359, GM2602, GM10277, FAM222A, FOXA3, AOC2, SERPINA1E, CTXN1, RAPGEF4OS2, SOCS2, PPAN, PRKAG2OS1, GADD45B, HOXA5, GRHL1, EIF4EBP3, OSGIN1, GM28513 , MAP3K6, SLC34A2, B630019A10RIK, IGKC, PLIN4, ANGPTL4, DUSP5, EGR1, GM42507, GM14257, APOLD1, IER3, ZBTB16, GM37033, IGLC1, GADD45G, IGLC3, GM expression of one or more genes in the liver of the subject selected from the group consisting of 45244, RGS1, CXCL1, RNF225, GM44005, ANKRD37, NR4A1, GM8893, GM26762, CDKN1A, 5330406M23RIK, IGLV1, IGKV3-2, FOS, GM43637, IGKV3-10, S100A9, GM15622, S100A8, MT1, RETNLG, MT2, IGKV19-93, GM45774, and SERPINA4-PS1, compared to the expression level of the one or more genes in the subject prior to administration;In some embodiments of any of the methods described herein, the administration results in a decrease in DBP, IGKV4-55, PER3, MUP-PS10, GPAM, TMPRSS4, MUP-PS14, AC166078.1, MUP-PS12, GM2065, A530020G20RIK, ACSS2OS, DCLK3, KLF12, GM44669, MFSD9, B4GALNT3, GM3776, TMEM167-PS1, KRT23, LMBRD2, GM22935, SULT2A-PS1, SNAI3, GM15908, MIR6392, ACSS2, NR1 D1, BC049987, CCDC85C, CES2C, ACPP, MUP2, PTK6, UGT1A5, 1810008I18RIK, IL22RA1, ACSS3, ADNP, RDH16, SNTB1, 4933411K16RIK, NTRK2, EXTL1, PSTPI P2, RASSF6, AQP4, UGT1A9, PROM1, ZFP608, FAM13A, NFE2, TEF, TNFAIP8L3, SCD1, MMD2, SYNE3, ACLY, C330021F23RIK, STON2, LRFN4, HHIPL1, WNT9B, NR1 D2, 1810049J17RIK, PDPR, NA, GM45884, SLC2A5, FAM83F, ZFP526, SGK2, GM43080, DEAF1, ME1, BMF, WDFY2, ADCY9, CLSTN3, ACOT11, LYST, LRTM1, OAT, VP S13C, E330011O21RIK, P2RY4, GM11437, RWDD2A, SVIL, ECHDC1, TRIM14, SLC10A5, TRHDE, MASP1, 2900097C17RIK, NDST1, RDH9, 1110002L01RIK, ABTB2, RGR, ACACB, SACM1L, DYRK2, ROBO1, GM44744, EIF4EBP2, KLHL24, CYP2A5, TIAM2, RAB43, GM13855, 9130409I23RIK, STON1, USP9X, UGT3A1, 9030616G12R IK, DOCK8, KLB, ACE, VLDLR, PCDHGC3, ABCA6, 4932422M17RIK, GM45838, FARP2, GM47205, SP4, UGT1A6B, KLHL28, D130043K22RIK, ASIC5, PM20D2, A1CF,SORBS1, SLC10A2, GM10642, UTP14B, GM38394, AFP, INSIG1, HNF1AOS2, METTL4, LSS, MTMR9, HMGCR, GDAP10, ADRA1A, ZFP773, CR KL, CHRNE, STARD13, CRY2, FADS2, COG5, FV1, RCAN2, ABCB1A, PPARA, ATP7A, MVD, 2610037D02RIK, TNFRSF14, SUCNR1, ECI3, ABC C4, LNCBATE1, MINDY2, BTBD7, 4933404O12RIK, ABCD1, FMN1, FNIP2, ABHD15, NKX2-6, C77080, GM43611, SGTB, ACSL3, NR5A2, FA M198A, KCTD7, ACACA, ZFP955B, SULT2A3, FZD4, FASN, CYP3A59, ZFP354B, TNFSF10, SESN3, MN1, RNF152, DHCR24, SPHK2, SYTL5, G M6652, BAHCC1, GAREM1, MFSD4A, HGF, GM3571, NOS1AP, DIXDC1, KANK1, REPS2, ASAH2, SEMA3B, RNF103, ZC3H12C, CDS2, DCUN1D4 , 2900026A02RIK, CYYR1, EEPD1, P2RY2, CYP2C39, SEC22C, EHHADH, ABCA3, HIPK2, RBM20, GRAMD4, FCHSD2, MOB3A, HMGN3, KLHDC7 A, VCP-RS, TERT, CYP3A41B, ARL13B, ZC3H12D, TLCD2, SNHG11, SORL1, GPR157, DNAJA4, TMEM253, TACO1, SPATA5L1, RHBG, COL15A1, PCDH12, IRS1, ASCC3, KIF16B, and MR1, resulting in an increase in the expression of one or more genes in the liver of a subject, compared to the expression level of the one or more genes in the subject before administration.

[0015] In some embodiments of any of the methods described herein, the subject has previously been identified or diagnosed as having liver disease or metabolic syndrome. In some embodiments of any of the methods described herein, the subject has previously been identified or diagnosed as having liver disease. In some embodiments of any of the methods described herein, the liver disease is selected from the group of fatty liver disease, hepatic steatosis, acute hepatic porphyria, Alagille syndrome, alcohol-related liver disease, alpha-1 antitrypsin deficiency, autoimmune hepatitis, benign liver tumors, cholangiocarcinoma, biliary atresia, Budd-Chiari syndrome, cirrhosis, Crigler-Najjar syndrome, galactosemia, Gilbert syndrome, hemochromatosis, hepatic encephalopathy, hepatitis A, hepatitis B, hepatitis C, hepatorenal syndrome, intrahepatic cholestasis of pregnancy (ICP), lysosomal acid lipase deficiency (LAL-D), liver cysts, liver cancer, neonatal jaundice, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis (PFIC), Reye's syndrome, type 1 glycogen storage disease, and Wilson's disease. In some embodiments of any of the methods described herein, the subject has previously been identified or diagnosed as having metabolic syndrome, which in some embodiments of any of the methods described herein is selected from the group consisting of coronary heart disease, pulmonary disease, gallbladder disease, dyslipidemia, hypertension, type 2 diabetes, dementia, cancer, gynecological disorders including polycystic ovary syndrome, osteoarthritis, pancreatitis, idiopathic intracranial hypertension, stroke, and cataracts.

[0016] In some embodiments of any of the methods described herein, the first target binding domain and the soluble tissue factor domain are immediately adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the soluble tissue factor domain and the first domain of the pair of affinity domains are immediately adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide in the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the pair of affinity domains.

[0017] In some embodiments of any of the methods described herein, the second domain of the pair of affinity domains and the second target binding domain are immediately adjacent to each other in the second chimeric polypeptide. In some embodiments of any of the methods described herein, the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target binding domain in the second chimeric polypeptide.

[0018] In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain are antigen binding domains. In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain are soluble interleukin or cytokine receptors.

[0019] In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises one or more additional target binding domains. In some embodiments of any of the methods described herein, the second chimeric polypeptide further comprises one or more additional target binding domains.

[0020] In some embodiments of any of the methods described herein, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments of any of the methods described herein, the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO: 1. In some embodiments of any of the methods described herein, the affinity domain pair is a sushi domain from the alpha chain of the human IL-15 receptor (IL-15Rα) and soluble IL-15.

[0021] In some embodiments of any of the methods described herein, the first target binding domain comprises soluble TGF-βRII. In some embodiments of any of the methods described herein, the first target binding domain comprises a first sequence at least 80% identical to SEQ ID NO:2 and a second sequence at least 80% identical to SEQ ID NO:2, wherein the first and second sequences are separated by a linker. In some embodiments of any of the methods described herein, the first target binding domain comprises a first sequence at least 90% identical to SEQ ID NO:2 and a second sequence at least 90% identical to SEQ ID NO:2. In some embodiments of any of the methods described herein, the first target binding domain comprises a first sequence of SEQ ID NO:2 and a second sequence of SEQ ID NO:2. In some embodiments of any of the methods described herein, the linker comprises the sequence of SEQ ID NO:3. In some embodiments of any of the methods described herein, the first target binding domain comprises a sequence at least 80% identical to SEQ ID NO:4. In some embodiments of any of the methods described herein, the first target binding domain comprises a sequence that is at least 90% identical to SEQ ID NO: 4. In some embodiments of any of the methods described herein, the first target binding domain comprises the sequence of SEQ ID NO: 4. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 6. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 6. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises the sequence of SEQ ID NO: 6. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises the sequence of SEQ ID NO: 7.

[0022] In some embodiments of any of the methods described herein, the second target binding domain comprises soluble TGF-βRII. In some embodiments of any of the methods described herein, the second target binding domain comprises a first sequence at least 80% identical to SEQ ID NO:2 and a second sequence at least 80% identical to SEQ ID NO:2, wherein the first and second sequences are separated by a linker. In some embodiments of any of the methods described herein, the second target binding domain comprises a first sequence at least 90% identical to SEQ ID NO:2 and a second sequence at least 90% identical to SEQ ID NO:2. In some embodiments of any of the methods described herein, the second target binding domain comprises a first sequence of SEQ ID NO:2 and a second sequence of SEQ ID NO:2. In some embodiments of any of the methods described herein, the linker comprises the sequence of SEQ ID NO:3. In some embodiments of any of the methods described herein, the second target binding domain comprises a sequence at least 80% identical to SEQ ID NO:4. In some embodiments of any of the methods described herein, the second target binding domain comprises a sequence at least 90% identical to SEQ ID NO:4. In some embodiments of any of the methods described herein, the second target binding domain comprises the sequence of SEQ ID NO:4. In some embodiments of any of the methods described herein, the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO:5. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO:6. In some embodiments of any of the methods described herein, the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO:5. In some embodiments of any of the methods described herein, the second chimeric polypeptide comprises the sequence of SEQ ID NO:5. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises the sequence of SEQ ID NO:6. In some embodiments of any of the methods described herein, the second chimeric polypeptide comprises the sequence of SEQ ID NO:8.

[0023] As used herein, the term "chimeric" refers to a polypeptide that includes amino acid sequences (e.g., domains) that are originally derived from two different sources (e.g., two different naturally occurring proteins, e.g., from the same species or different species). For example, a chimeric polypeptide can include domains that are derived from at least two different naturally occurring human proteins. In some examples, a chimeric polypeptide can include a domain that is a synthetic sequence (e.g., an scFv) and a domain that is derived from a naturally occurring protein (e.g., a naturally occurring human protein). In some embodiments, a chimeric polypeptide can include at least two different domains that are synthetic sequences (e.g., two different scFvs).

[0024] An "antigen-binding domain" is one or more protein domains (e.g., formed from amino acids from a single polypeptide, or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides)) that can specifically bind to one or more different antigens. In some examples, an antigen-binding domain can bind to an antigen or epitope with specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, the antigen-binding domain may be an antibody or fragment thereof. In some embodiments, the antigen-binding domain may comprise an alternative scaffold. Non-limiting examples of antigen-binding domains are described herein. Further examples of antigen-binding domains are known in the art.

[0025] A "soluble tissue factor domain" refers to a polypeptide having at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity) to a segment of a wild-type mammalian tissue factor protein (e.g., a wild-type human tissue factor protein) lacking the transmembrane and intracellular domains. Non-limiting examples of soluble tissue factor domains are described herein.

[0026] The term "soluble interleukin receptor" is used herein in the broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) that is capable of binding to one or more of its natural ligands (e.g., under physiological conditions, e.g., in phosphate-buffered saline at room temperature). For example, a soluble interleukin receptor can include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the extracellular domain of a wild-type interleukin receptor and retains its ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally further lacks its intracellular domain). Non-limiting examples of soluble interleukin receptors are described herein.

[0027] The term "soluble cytokine receptor" is used herein in the broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) that is capable of binding to one or more of its natural ligands (e.g., under physiological conditions, e.g., in phosphate-buffered saline at room temperature). For example, a soluble cytokine receptor can comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the extracellular domain of a wild-type cytokine receptor and retains its ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally further lacks its intracellular domain). Non-limiting examples of soluble cytokine receptors are described herein.

[0028] The term "antibody" is used herein in its broadest sense and includes a specific type of immunoglobulin molecule that contains one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. An example of an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer. Further examples of antibodies are described herein. Further examples of antibodies are known in the art.

[0029] "Affinity" refers to the strength of the sum of non-covalent interactions between an antigen-binding site and its binding partner (e.g., antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between a member of the antigen-binding domain and an antigen or epitope. The affinity of a molecule X for its partner Y is determined by the dissociation equilibrium constant (K D ) The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®). Additional methods for determining the affinity of antigen-binding domains and their corresponding antigens or epitopes are known in the art.

[0030] A "multi-chain polypeptide," as used herein, refers to a polypeptide comprising two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) protein chains (e.g., at least a first chimeric polypeptide and a second polypeptide), wherein the two or more protein chains associate via non-covalent bonds to form a quaternary structure.

[0031] The term "affinity domain pair" refers to a pair of affinity domains with a density of 1 × 10 -7 K less than M D (e.g., 1×10-8 Less than M, 1 x 10 -9 Less than M, 1 x 10 -10 Less than M or 1 x 10 -11 Affinity domain pairs are two different protein domains that specifically bind to each other with a specific affinity (M or less). In some examples, an affinity domain pair can be a pair of naturally occurring proteins. In some embodiments, an affinity domain pair can be a pair of synthetic proteins. Non-limiting examples of affinity domain pairs are described herein.

[0032] The term "epitope" refers to the portion of an antigen that specifically binds to an antigen-binding domain. An epitope may consist of, for example, surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter may be lost in the presence of denaturing solvents. An epitope may include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding. Methods for identifying the epitope to which an antigen-binding domain binds are known in the art.

[0033] "Immune effector cell" refers to a cell of a mammalian immune system that can directly or indirectly recognize and / or cause cytostasis or cell death of pathogenic cells (e.g., cancer cells) in a mammal. Non-limiting examples of immune effector cells include macrophages, T lymphocytes (e.g., cytotoxic T lymphocytes and T helper cells), natural killer cells, neutrophils, monocytes, and eosinophils. Further examples of immune effector cells are known in the art.

[0034] The term "treatment" means ameliorating at least one symptom of a disorder. In some instances, the disorder being treated is cancer, and ameliorating at least one symptom of cancer involves reducing the abnormal proliferation, gene expression, signal transduction, translation, and / or secretion of a factor. Generally, treatment methods involve administering to a subject in need of, or determined to be in need of, such treatment a therapeutically effective amount of a composition that alleviates at least one symptom of the disorder.

[0035] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein. Other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will govern.

[0036] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0037] [Figure 1] Figure 1 shows an exemplary diagram of a multi-chain chimeric polypeptide: (i) a first chimeric polypeptide comprising a first target-binding domain (A), a soluble tissue factor domain, a first domain of an affinity pair of domains (soluble interleukin IL-15), and an additional target-binding domain (B); and (ii) a second chimeric polypeptide comprising a second domain of an affinity pair of domains (IL-15 receptor alpha sushi domain), a second target-binding domain (C), and an additional antigen-binding domain (D). The top schematic shows the association of the first and second chimeric polypeptides via the affinity domain pair. The bottom schematic shows the order of the domains in the first and second chimeric polypeptides. [Figure 2]

[0033] Figure 1 shows exemplary diagrams of multi-chain chimeric polypeptides: (i) a first chimeric polypeptide comprising a first target-binding domain (A), a soluble tissue factor domain comprising five amino acid substitutions to eliminate binding of the soluble tissue factor domain to FVIIa, a first domain of an affinity pair of domains (soluble interleukin IL-15 comprising D8N or D8A amino acid substitutions), and an additional target-binding domain (B); and (ii) a second chimeric polypeptide comprising a second domain of an affinity pair of domains (IL-15 receptor alpha sushi domain), a second target-binding domain (C), and an additional antigen-binding domain (D). The top schematic shows the association of the first and second chimeric polypeptides via the affinity domain pair. The bottom schematic shows the order of the domains in the first and second chimeric polypeptides. In other embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain can comprise or consist of a soluble wild-type human tissue factor domain (comprising or consisting of a contiguous sequence within wild-type human tissue factor). [Figure 3] A schematic diagram of the TGFRt15-TGFRs construct is shown. [Figure 4] A further schematic diagram of the TGFRt15-TGFRs construct is shown. [Figure 5] 1 shows the results of TGFβ1 inhibition by TGFRt15-TGFRs and TGFR-Fc. [Figure 6] 1 shows the results of a 32D β cell proliferation assay using TGFRt15-TGFRs or recombinant IL-15. [Figure 7A] 7A and 7B show the results of ELISA detection of IL-15 and TGFβRII in TGFRt15-TGFRs using the corresponding antibodies. [Figure 7B] See legend to Figure 7A. [Figure 8] 1 is a line graph showing the chromatographic profile of TGFRt15-TGFRs protein-containing cell culture supernatant after binding on an anti-TF antibody resin and elution. [Figure 9]1 shows the analytical SEC profile of TGFRt15-TGFRs. [Figure 10] Shown are TGFRt15-TGFRs before and after deglycosylation analyzed by reducing SDS-PAGE. [Figure 11A] Figures 11A and 11B show spleen weights and percentages of immune cell types in TGFRt15-TGFRs-treated and control-treated mice. Figure 85A shows spleen weights in mice treated with TGFRt15-TGFRs compared to PBS controls. Figure 85B shows percentages of CD4+ T cells, CD8+ T cells, and NK cells in mice treated with TGFRt15-TGFRs compared to PBS controls. [Figure 11B] See legend to Figure 11A. [Figure 12A] Figures 12A and 12B show spleen weight and immune stimulation over 92 hours in mice treated with TGFRtl5-TGFRs. Figure 86A shows spleen weight in mice treated with TGFRtl5-TGFRs at 16, 24, 48, 72, and 92 hours after treatment. Figure 86B shows the percentage of immune cells in mice treated with TGFRtl5-TGFRs at 16, 24, 48, 72, and 92 hours after treatment. [Figure 12B] See legend to Figure 12A. [Figure 13A] 13A and 13B show Ki67 and Granzyme B expression over time in mice treated with TGFRt15-TGFRs. [Figure 13B] See legend to Figure 13A. [Figure 14] 1 shows enhanced cytotoxicity of splenocytes by TGFRt15-TGFRs in C57BL / 6 mice. [Figure 15] 1 shows the change in tumor size in response to PBS treatment, chemotherapy alone, TGFRt15-TGFRs alone, or a combination of chemotherapy and TGFRt15-TGFRs in a mouse model of pancreatic cancer. [Figure 16] 1 shows the cytotoxicity of NK cells isolated from mice treated with TGFRt15-TGFRs. [Figure 17A]17A-17C show in vivo stimulation of Tregs, NK cells, and CD8+ T cells in ApoE- / - mice fed a Western diet and treated with TGFRt15-TGFRs. [Figure 17B] See legend to Figure 17A. [Figure 17C] See legend to Figure 17A. [Figure 18A] 18A-18C show immune stimulation in C57BL / 6 mice after treatment with TGFRt15-TGFRs. [Figure 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 19A] 19A and 19B show in vivo induction of NK cell and CD8+ T cell proliferation in ApoE- / - mice fed a Western diet and treated with TGFRt15-TGFRs. [Figure 19B] See legend to Figure 19A. [Figure 20A] Figures 20A and 20B show the enhancement of NK cell cytotoxicity after treatment of NK cells with TGFRt15-TGFRs. [Figure 20B] See legend to Figure 20A. [Figure 21A] 21A and 21B show the enhancement of ADCC activity of NK cells after treatment of the NK cells with TGFRt15-TGFRs. [Figure 21B] See legend to Figure 21A. [Figure 22A] 22A-22H show the anti-cancer activity of TGFRt15-TGFRs+anti-TRP1(TA99) in combination with chemotherapy in a melanoma mouse model. [Figure 22B] See legend to Figure 22A. [Figure 22C] See legend to Figure 22A. [Figure 22D] See legend to Figure 22A. [Figure 22E] See legend to Figure 22A. [Figure 22F]See legend to Figure 22A. [Figure 22G] See legend to Figure 22A. [Figure 22H] See legend to Figure 22A. [Figure 23A] 23A-23C show amelioration of Western diet-induced hyperglycemia in ApoE− / − mice by TGFRt15-TGFRs. [Figure 23B] See legend to Figure 22A. [Figure 23C] See legend to Figure 22A. [Figure 24] Figure 1 shows upregulation of CD44hi memory T cells upon treatment with TGFRt15-TGFRs. [Figure 25] 1 shows RNA-seq analysis of differentially expressed genes between PBS (control group) or TGFRt15-TGFRs (TGFRt15-TGFRs group) in the liver of db / db mice. [Figure 26] 1 shows RNA-seq analysis of differentially expressed genes between PBS (control group) or TGFRt15-TGFRs (TGFRt15-TGFRs group) in aged mouse liver. [Figure 27] Volcano plot of RNA-seq analysis of db / db mouse liver. [Figure 28] A heatmap depicting differentially expressed genes as determined by RNA-seq analysis of livers from db / db mice treated with TGFRt15-TGFRs (HCW9218) and PBS negative control is shown. [Figure 29] A heatmap of differentially expressed senescence- and inflammation-related genes measured by RNA-seq in the livers of aged mice is shown. [Figure 30] FIG. 1 shows a schematic diagram of the study design to investigate TGFRt15-TGFRs (HCW9218) treatment in the db / db mouse model. [Figure 31]Relative mRNA expression of IL1β, IL1α, PAI-1, IL6, and Tnfα in the liver measured by quantitative PCR after treatment with TGFRt15-TGFRs (HCW9218) compared to controls on days 10 or 60 is shown. [Figure 32] Figure 1 shows the relative mRNA expression of IL1β, IL1α, PAI-1, IL6, and Cdkn1a in the liver after treatment with one or two TGFRt15-TGFRs (HCW9218) doses compared to control at day 120, as measured by quantitative PCR. [Figure 33] ELISA data for IL-1α, IL-6, and IL-8 protein levels in liver tissue after treatment with TGFRt15-TGFRs (HCW9218) compared to controls after 120 days of treatment are shown. [Figure 34] ELISA data for PAI-1 and fibronectin protein levels in liver tissue after treatment with TGFRt15-TGFRs (HCW9218) compared to controls after 120 days of treatment are shown. [Figure 35] Immunofluorescence staining of p21+ expressing liver tissue cells after treatment with two doses of TGFRt15-TGFRs (HCW9218) compared to PBS negative control. [Figure 36] Shown is a heatmap of differentially expressed genes detected by RNA-seq data generated from livers of aged mice receiving either PBS only or negative control TGFRt15-TGFRs (HCW9218) treatment. [Figure 37] 1 shows flow cytometry analysis of Ki67 expression on CD4, CD8, Treg and CD16+ NK cells in blood from cynomolgus monkeys after treatment with TGFRt15-TGFRs (HCW9218). [Figure 38] 1 shows flow cytometry analysis of absolute numbers of CD4, CD8, Treg, and CD16+ NK cells in blood from cynomolgus monkeys after treatment with TGFRt15-TGFRs (HCW9218). [Figure 39]1 shows that TGFRt15-TGFRs treatment enhances immune cell populations in db / db mice. [Figure 40] 1 shows the effect of treatment with TGFRt15-TGFRs or TGFRt15*-TGFRs on the cytotoxic activity of splenocytes in db / db mice on day 4 after treatment. [Figure 41] 1 shows the effect of TGFRt15-TGFRs and TGFRt15*-TGFRs treatment on interferon-γ production by splenocytes in db / db mice on day 4 after treatment and in vitro αCD3 / CD28 stimulation assay. [Figure 42] 1 shows the effect of TGFRt15-TGFRs on glycolytic activity of splenocytes in db / db mice 4 days after treatment. [Figure 43] 1 shows the effect of TGFRt15-TGFRs on mitochondrial respiration of splenocytes in db / db mice 4 days after treatment. [Figure 44] 1 shows the effect of TGFRt15-TGFRs on plasma TGFβ1 and TGFβ2 levels in db / db mice after 4 days of treatment. [Figure 45] Figure 1 shows the effect of TGFRt15-TGFRs (HCW9218) on protecting against chemically induced liver injury. DETAILED DESCRIPTION OF THE INVENTION

[0038] Detailed Description Nonalcoholic fatty liver disease (NAFLD) has emerged as a major chronic liver disease worldwide, affecting an estimated 1 billion people worldwide (Younossi et al., Hepatology 69(6):2672-2682, 2019). NAFLD represents a spectrum of liver diseases ranging from isolated steatosis (NAFL) to nonalcoholic steatohepatitis (NASH), fibrotic NASH, progressive fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) (Meijnikman et al., JHEP Rep. 3(4):100301, 2021).

[0039] The metabolic mechanisms leading to NAFLD reflect an imbalance in energy metabolism in the liver. The liver's inability to oxidize excess energy, primarily in the form of carbohydrates and fats, to CO2 or to export it as very-low-density lipoproteins (VDL). This results in the net accumulation of energy in the liver as triglycerides, which explains the widespread presence of NAFLD in obese individuals and those with lipodystrophy. Skeletal muscle insulin resistance, one of the earliest defects associated with metabolic syndrome and prediabetes, can also promote the development of NAFLD through increased hepatic de novo lipogenesis (DNL) and hypertriglyceridemia by diverting ingested glucose from skeletal muscle glycogen synthesis to the liver for DNL. The development of hepatic insulin resistance, which impairs insulin activation of glycogen synthase (Loomba et al., Cell 184(10):2537-2564, 2021), redirects glucose into the lipogenic pathway, further promoting NAFLD.

[0040] Adipocyte dysfunction also promotes the development of NAFLD. Severe NAFLD / NASH is a complication of congenital lipodystrophy, in which the lack of adipose tissue causes the liver to store excess fatty acids, resulting in severe insulin resistance. Other metabolic causes of fatty liver include (1) defective intrahepatic lipolysis, (2) defective triglyceride transport, (3) increased glucokinase activity leading to hepatic DNL, ​​and (4) decreased hepatic mitochondrial / peroxisomal β-oxidation (Loomba et al., Cell 184(10):2537-2564, 2021).

[0041] Hepatocytes begin to accumulate fat as they synthesize new lipids via the DNL pathway, and adaptive responses counteract the production of toxic lipid metabolites and balance excess free fatty acids (Piccinin et al., Nat. Rev. Gastroenterol. Hepatol. 16(3):160-174, 2019). Accumulated toxic metabolites promote an inflammatory state in the liver, which is further exacerbated by endotoxins derived from increased intestinal permeability and dysbiosis, as well as the release of IL-6 and TNF from inflamed adipose tissue (Loomba et al., Cell 184(10):2537-2564, 2021; Yki-Jarvinen et al., Nat. Rev. Gastroenterol. Hepatol. 2021). Therefore, the hepatic inflammatory microenvironment plays a key role in the development of NAFLD and its progression to HCC. Furthermore, cytokine-mediated hepatocyte injury and death are followed by proliferation of hepatic progenitor cell populations, which, in an inflammatory environment, induce a fibrogenic response in hepatic stellate cells, thereby promoting liver fibrosis and progression to NASH (Loomba et al., Cell 184(10):2537-2564, 2021). Recent evidence suggests that hepatocyte senescence is also a causative factor in the development of NAFLD.

[0042] NAFLD is associated with several markers of aging in hepatocytes, such as increased senescence-associated lesions, increased senescence-associated dilation, and larger nuclear area (Ogrodnik et al., Nat. Comm. 8:15691, 2017). Hepatocyte senescence has also been shown to impair hepatic mitochondrial β-oxidation, thereby impairing fatty acid clearance and promoting triglyceride accumulation (Ogrodnik et al., Nat. Comm. 8:15691, 2017). The paracrine effects of pro-inflammatory factors secreted from the senescence-associated secretory phenotype of senescent hepatocytes disrupt the normal metabolism of healthy hepatocytes (Loomba et al., Cell 184(10):2537-2564, 2021). Finally, the causal relationship between hepatocellular senescence and hepatic steatosis was elucidated using transgenic mice and a senolytic cocktail (Childs et al., Nat. Rev. Drug Discov. 16(10):718-735, 2017).

[0043] Herein, we demonstrate that subcutaneous administration of TGFRt15-TGFRs can eliminate hepatocyte senescence, reduce the inflammatory microenvironment in the liver, rebalance liver metabolic function, and reduce gluconeogenesis and lipogenesis in Db / Db and natural aging mouse models. Therefore, TGFRt15-TGFRs has the potential to treat various liver diseases and metabolic syndrome.

[0044] Methods for treating liver disease or metabolic syndrome in a subject diagnosed with liver disease or metabolic syndrome; methods for reducing one or more of the rate of progression from non-alcoholic fatty liver disease (NAFL) to non-alcoholic steatohepatitis (NASH), from NASH to cirrhosis, and from cirrhosis to hepatocellular carcinoma; methods for reducing inflammation in the liver of a subject identified as being in need thereof; methods for reducing gluconeogenesis in the liver of a subject identified as being in need thereof; methods for reducing lipogenesis in the liver of a subject identified as being in need thereof; methods for reducing hepatocyte senescence in the liver of a subject identified as being in need thereof; methods for rebalancing metabolic function in the liver of a subject identified as being in need thereof; and methods for reducing inflammation in the liver of a subject identified as being in need thereof.

[0013] Provided herein is a method of modulating expression of one or more genes in Tables 1-4 in the liver of a subject identified as being in need thereof, the method comprising administering a therapeutically effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising: (i) a second domain of the pair of affinity domains, and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through binding of the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII.

[0045] In some examples of any of the multi-chain chimeric polypeptides described herein, the total length of the first chimeric polypeptide and / or the second chimeric polypeptide is, independently, from about 50 amino acids to about 3000 amino acids, from about 50 amino acids to about 2500 amino acids, from about 50 amino acids to about 2000 amino acids, from about 50 amino acids to about 1500 amino acids, from about 50 amino acids to about 1000 amino acids, from about 50 amino acids to about 950 amino acids, from about 50 amino acids to about 900 amino acids, from about 50 amino acids to about 850 amino acids, from about 50 amino acids to about 800 amino acids, from about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 480 amino acids, about 50 amino acids to about 460 amino acids, about 50 amino acids to about 440 amino acids, about 50 amino acids to about 420 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 380 amino acids, about 50 amino acids to about 360 amino acids, about 50 amino acids to about 340 amino acids, about 50 amino acids to about 320 amino acids, about 50 Amino acids up to about 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 3000 amino acids, about 100 amino acids to about 2500 amino acids, about 100 amino acids to about 2000 amino acids, about 100 amino acids to about 1500 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 950 amino acids, about 10 0 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 480 amino acids, about 100 amino acids to about 460 amino acids, about 100 amino acids to about 440 amino acids, about 100 amino acids to about 420 amino acids, about 100 amino acids to about 400 amino acids,About 100 amino acids to about 380 amino acids, about 100 amino acids to about 360 amino acids, about 100 amino acids to about 340 amino acids, about 100 amino acids to about 320 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 280 amino acids, about 100 amino acids to about 260 amino acids, about 100 amino acids to about 240 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 3000 amino acids, about 150 amino acids to about 2500 amino acids, about 150 amino acids to about 2000 Amino acids, about 150 amino acids to about 1500 amino acids, about 150 amino acids to about 1000 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids to about 850 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 480 amino acids, about 150 amino acids to about About 460 amino acids, about 150 to about 440 amino acids, about 150 to about 420 amino acids, about 150 to about 400 amino acids, about 150 to about 380 amino acids, about 150 to about 360 amino acids, about 150 to about 340 amino acids, about 150 to about 320 amino acids, about 150 to about 300 amino acids, about 150 to about 280 amino acids, about 150 to about 260 amino acids, about 150 to about 240 amino acids, about 150 to about 220 amino acids, about 150 to about 200 amino acids, about 200 amino acids amino acids to about 3000 amino acids, about 200 amino acids to about 2500 amino acids, about 200 amino acids to about 2000 amino acids, about 200 amino acids to about 1500 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids,About 200 to about 500 amino acids, about 200 to about 480 amino acids, about 200 to about 460 amino acids, about 200 to about 440 amino acids, about 200 to about 420 amino acids, about 200 to about 400 amino acids, about 200 to about 380 amino acids, about 200 to about 360 amino acids, about 200 to about 340 amino acids, about 200 to about 320 amino acids, about 200 to about 300 amino acids, about 200 to about 280 amino acids, about 200 to about 260 amino acids, about 200 to about 240 amino acids amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 3000 amino acids, about 220 amino acids to about 2500 amino acids, about 220 amino acids to about 2000 amino acids, about 220 amino acids to about 1500 amino acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220 amino acids to about 900 amino acids, about 220 amino acids to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750 amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 amino acids, about 220 amino acids to about About 600 amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to about 500 amino acids, about 220 amino acids to about 480 amino acids, about 220 amino acids to about 460 amino acids, about 220 amino acids to about 440 amino acids, about 220 amino acids to about 420 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 380 amino acids, about 220 amino acids to about 360 amino acids, about 220 amino acids to about 340 amino acids, about 220 amino acids to about 320 amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to about 280 amino acids, about 220 amino acids amino acids to about 260 amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to about 3000 amino acids, about 240 amino acids to about 2500 amino acids, about 240 amino acids to about 2000 amino acids, about 240 amino acids to about 1500 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids,About 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids, about 240 amino acids to about 500 amino acids, about 240 amino acids to about 480 amino acids, about 240 amino acids to about 460 amino acids, about 240 amino acids to about 440 amino acids, about 240 amino acids to about 420 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 380 amino acids, about 240 amino acids to about 360 amino acids, about 240 amino acids to about 340 amino acids, about 240 amino acids to about 320 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids amino acids, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 3000 amino acids, about 260 amino acids to about 2500 amino acids, about 260 amino acids to about 2000 amino acids, about 260 amino acids to about 1500 amino acids, about 260 amino acids to about 1000 amino acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about About 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 480 amino acids, about 260 amino acids to about 460 amino acids, about 260 amino acids to about 440 amino acids, about 260 amino acids to about 420 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 380 amino acids, about 260 amino acids to about 360 amino acids, about 260 amino acids to about 340 amino acids, about 260 amino acids to about 320 amino acids, about 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids amino acids to about 3000 amino acids, about 280 amino acids to about 2500 amino acids, about 280 amino acids to about 2000 amino acids, about 280 amino acids to about 1500 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids,About 280 amino acids to about 500 amino acids, about 280 amino acids to about 480 amino acids, about 280 amino acids to about 460 amino acids, about 280 amino acids to about 440 amino acids, about 280 amino acids to about 420 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 380 amino acids, about 280 amino acids to about 360 amino acids, about 280 amino acids to about 340 amino acids, about 280 amino acids to about 320 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 3000 amino acids, about 300 amino acids to about 2500 amino acids, about 300 amino acids to about 2000 Amino acids, about 300 to about 1500 amino acids, about 300 to about 1000 amino acids, about 300 to about 950 amino acids, about 300 to about 900 amino acids, about 300 to about 850 amino acids, about 300 to about 800 amino acids, about 300 to about 750 amino acids, about 300 to about 700 amino acids, about 300 to about 650 amino acids, about 300 to about 600 amino acids, about 300 to about 550 amino acids, about 300 to about 500 amino acids, about 300 to about 480 amino acids, about 300 to about About 460 amino acids, about 300 to about 440 amino acids, about 300 to about 420 amino acids, about 300 to about 400 amino acids, about 300 to about 380 amino acids, about 300 to about 360 amino acids, about 300 to about 340 amino acids, about 300 to about 320 amino acids, about 320 to about 3000 amino acids, about 320 to about 2500 amino acids, about 320 to about 2000 amino acids, about 320 to about 1500 amino acids, about 320 to about 1000 amino acids, about 320 to about 950 amino acids, about 3 20 amino acids to about 900 amino acids, about 320 amino acids to about 850 amino acids, about 320 amino acids to about 800 amino acids, about 320 amino acids to about 750 amino acids, about 320 amino acids to about 700 amino acids, about 320 amino acids to about 650 amino acids, about 320 amino acids to about 600 amino acids, about 320 amino acids to about 550 amino acids, about 320 amino acids to about 500 amino acids, about 320 amino acids to about 480 amino acids, about 320 amino acids to about 460 amino acids, about 320 amino acids to about 440 amino acids, about 320 amino acids to about 420 amino acids, about 320 amino acids to about 400 amino acids,About 320 amino acids to about 380 amino acids, about 320 amino acids to about 360 amino acids, about 320 amino acids to about 340 amino acids, about 340 amino acids to about 3000 amino acids, about 340 amino acids to about 2500 amino acids, about 340 amino acids to about 2000 amino acids, about 340 amino acids to about 15, 00 amino acids, about 340 amino acids to about 1000 amino acids, about 340 amino acids to about 950 amino acids, about 340 amino acids to about 900 amino acids, about 340 amino acids to about 850 amino acids, about 340 amino acids to about 800 amino acids, about 340 amino acids to about 750 amino acids, about 340 amino acids to about 700 amino acids, about 340 amino acids to about 650 amino acids, about 340 amino acids to about 600 amino acids, about 340 amino acids to about 550 amino acids, about 340 amino acids to about 500 amino acids, about 340 amino acids to about 480 amino acids, about 340 amino acids to about 460 amino acids, about 340 amino acids to about 500 amino acids amino acids to about 440 amino acids, about 340 amino acids to about 420 amino acids, about 340 amino acids to about 400 amino acids, about 340 amino acids to about 380 amino acids, about 340 amino acids to about 360 amino acids, about 360 amino acids to about 3000 amino acids, about 360 amino acids to about 2500 amino acids, about 360 amino acids to about 2000 amino acids, about 360 amino acids to about 1500 amino acids, about 360 amino acids to about 1000 amino acids, about 360 amino acids to about 950 amino acids, about 360 amino acids to about 900 amino acids, about 360 amino acids to about 850 amino acids, about 360 amino acids to about 800 amino acids amino acids, about 360 amino acids to about 750 amino acids, about 360 amino acids to about 700 amino acids, about 360 amino acids to about 650 amino acids, about 360 amino acids to about 600 amino acids, about 360 amino acids to about 550 amino acids, about 360 amino acids to about 500 amino acids, about 360 amino acids to about 480 amino acids, about 360 amino acids to about 460 amino acids, about 360 amino acids to about 440 amino acids, about 360 amino acids to about 420 amino acids, about 360 amino acids to about 400 amino acids, about 360 amino acids to about 380 amino acids, about 380 amino acids to about 3000 amino acids, about 380 amino acids to about 2500 amino acids, about 380 amino acids to about 2000 amino acids, about 380 amino acids to about 1500 amino acids, about 380 amino acids to about 1000 amino acids, about 380 amino acids to about 950 amino acids, about 380 amino acids to about 900 amino acids, about 380 amino acids to about 850 amino acids, about 380 amino acids to about 800 amino acids, about 380 amino acids to about 750 amino acids, about 380 amino acids to about 700 amino acids, about 380 amino acids to about 650 amino acids, about 380 amino acids to about 600 amino acids, about 380 amino acids to about 550 amino acids, about 380 amino acids to about 500 amino acids,About 380 amino acids to about 480 amino acids, about 380 amino acids to about 460 amino acids, about 380 amino acids to about 440 amino acids, about 380 amino acids to about 420 amino acids, about 380 amino acids to about 400 amino acids, about 400 amino acids to about 3000 amino acids, about 400 amino acids to about 2500 amino acids, about 400 amino acids to about 2000 amino acids, about 400 amino acids to about 1500 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 480 amino acids, about 400 amino acids to about 460 amino acids, about 400 amino acids to about 440 amino acids, about 400 amino acids to about 420 amino acids, about 420 amino acids to about 3000 amino acids, about 420 amino acids to about 2500 amino acids, about 420 amino acids to about 2000 amino acids , about 420 amino acids to about 1500 amino acids, about 420 amino acids to about 1000 amino acids, about 420 amino acids to about 950 amino acids, about 420 amino acids to about 900 amino acids, about 420 amino acids to about 850 amino acids, about 420 amino acids to about 800 amino acids, about 420 amino acids to about 750 amino acids, about 420 amino acids to about 700 amino acids, about 420 amino acids to about 650 amino acids, about 420 amino acids to about 600 amino acids, about 420 amino acids to about 550 amino acids, about 420 amino acids to about 500 amino acids, about 420 amino acids to about 480 amino acids, about 420 amino acids to about 460 amino acids, about 420 amino acids to about 440 amino acids, about 440 amino acids to about 3000 amino acids, about 440 amino acids to about 2500 amino acids, about 440 amino acids to about 2000 amino acids, about 440 amino acids to about 1500 amino acids, about 440 amino acids to about 1000 amino acids, about 440 amino acids to about 950 amino acids, about 440 amino acids to about 900 amino acids, about 440 amino acids to about 850 amino acids, about 440 amino acids to about 800 amino acids, about 440 amino acids to about 750 amino acids, about 440 amino acids to about 700 amino acids, about 440 amino acids to about 650 amino acids,About 440 amino acids to about 600 amino acids, about 440 amino acids to about 550 amino acids, about 440 amino acids to about 500 amino acids, about 440 amino acids to about 480 amino acids, about 440 amino acids to about 460 amino acids, about 460 amino acids to about 3000 amino acids, about 460 amino acids to about 2500 amino acids, about 460 amino acids to about 2000 amino acids, about 460 amino acids to about 1500 amino acids, about 460 amino acids to about 1000 amino acids, about 460 amino acids to about 950 amino acids, about 460 amino acids to about 900 amino acids, about 460 amino acids to about 850 amino acids, about 460 amino acids up to about 800 amino acids, about 460 amino acids to about 750 amino acids, about 460 amino acids to about 700 amino acids, about 460 amino acids to about 650 amino acids, about 460 amino acids to about 600 amino acids, about 460 amino acids to about 550 amino acids, about 460 amino acids to about 500 amino acids, about 460 amino acids to about 480 amino acids, about 480 amino acids to about 3000 amino acids, about 480 amino acids to about 2500 amino acids, about 480 amino acids to about 2000 amino acids, about 480 amino acids to about 1500 amino acids, about 480 amino acids to about 1000 amino acids, about 480 amino acids to about 950 amino acids , about 480 amino acids to about 900 amino acids, about 480 amino acids to about 850 amino acids, about 480 amino acids to about 800 amino acids, about 480 amino acids to about 750 amino acids, about 480 amino acids to about 700 amino acids, about 480 amino acids to about 650 amino acids, about 480 amino acids to about 600 amino acids, about 480 amino acids to about 550 amino acids, about 480 amino acids to about 500 amino acids, about 500 amino acids to about 3000 amino acids, about 500 amino acids to about 2500 amino acids, about 500 amino acids to about 2000 amino acids, about 500 amino acids to about 1500 amino acids, about 500 amino acids to about 1 about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 550 amino acids, about 550 amino acids to about 3000 amino acids, about 550 amino acids to about 2500 amino acids, about 550 amino acids to about 2000 amino acids, about 550 amino acids to about 1500 amino acids,About 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 3000 amino acids, about 600 amino acids to about 2500 amino acids, about 600 amino acids to about 2000 amino acids, about 600 amino acids to about 1500 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 3000 amino acids, about 650 amino acids to about 2500 amino acids, about 650 amino acids to about 2000 amino acids, about 650 amino acids to about 1500 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 3000 amino acids, about 700 amino acids to about 2500 amino acids, about 700 amino acids to about 2000 amino acids, about 700 amino acids to about 1500 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 8 00 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 3000 amino acids, about 750 amino acids to about 2500 amino acids, about 750 amino acids to about 2000 amino acids, about 750 amino acids to about 1500 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids, about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 3000 amino acids, about 800 amino acids to about 2500 amino acids, about 800 amino acids to about 2000 amino acids,About 800 amino acids to about 1500 amino acids, about 800 amino acids to about 1000 amino acids, about 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 3000 amino acids, about 850 amino acids to about 2500 amino acids, about 850 amino acids to about 2000 amino acids, about 850 amino acids to about 1500 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 3000 amino acids, about 900 amino acids to about 2500 amino acids, about 900 amino acids to about 2000 amino acids, about 900 amino acids to about 1500 amino acids, about 900 amino acids to about 1000 amino acids, about The length may be 900 to about 950 amino acids, about 950 to about 3000 amino acids, about 950 to about 2500 amino acids, about 950 to about 2000 amino acids, about 950 to about 1500 amino acids, about 950 to about 1000 amino acids, about 1000 to about 3000 amino acids, about 1000 to about 2500 amino acids, about 1000 to about 2000 amino acids, about 1000 to about 1500 amino acids, about 1500 to about 3000 amino acids, about 1500 to about 2500 amino acids, about 1500 to about 2000 amino acids, about 2000 to about 3000 amino acids, about 2000 to about 2500 amino acids, or about 2500 to about 3000 amino acids. Diagrams of exemplary multi-chain chimeric polypeptides provided herein are shown in Figures 1 and 2.

[0046] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the first target binding domains described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) are immediately adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or exemplary linker sequences known in the art) between the first target binding domain (e.g., any of the exemplary first target binding domains described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) in the first chimeric polypeptide.

[0047] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary affinity domain pairs described herein) are immediately adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary affinity domain pairs described herein) in the first chimeric polypeptide.

[0048] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second domain of the pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein) are immediately adjacent to each other in the second chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein) between the second domain of the pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein) in the second chimeric polypeptide.

[0049] Non-limiting aspects of these chimeric polypeptides, nucleic acids, vectors, cells, and methods are described below and can be used in any combination without limitation. Additional aspects of these chimeric polypeptides, nucleic acids, vectors, cells, and methods are known in the art.

[0050] tissue factor Human tissue factor is a 263-amino acid transmembrane protein containing three domains: (1) a 219-amino acid N-terminal extracellular domain (residues 1–219); (2) a 22-amino acid transmembrane domain (residues 220–242); and (3) a 21-amino acid cytoplasmic C-terminal tail (residues 242–263) (UniProtKB Identification Number: P13726). The cytoplasmic tail contains two phosphorylation sites at Ser253 and Ser258 and one S-palmitoylation site at Cys245. Deletion or mutation of the cytoplasmic domain has been found to have no effect on tissue factor clotting activity. Tissue factor has one S-palmitoylation site in the intracellular domain of the protein, at Cys245. Cys245 is located at the amino acid terminal of the intracellular domain, close to the membrane surface. The tissue factor transmembrane domain is composed of a single α-helix.

[0051] The extracellular domain of tissue factor, consisting of two fibronectin type III domains, is connected to the transmembrane domain via a six-amino acid linker. This linker provides conformational flexibility to separate the tissue factor extracellular domain from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module consists of two overlapping β-sheets, with an upper sheet domain containing three antiparallel β-strands and a lower sheet containing four β-strands. The β-strands are connected by β-loops between strands βA and βB, βC and βD, and βE and βF, all of which have conserved conformations in the two modules. Three short α-helical segments connect the β-strands. A unique feature of tissue factor is the 17-amino acid β-hairpin between strands β10 and β11, which is not a common element of the fibronectin superfamily. The N-terminal domain also contains a 12-amino acid loop between β6F and β7G, which is absent in the C-terminal domain and is unique to tissue factor. This fibronectin type III domain structure is characteristic of the immunoglobulin-like family of protein folds and is conserved among a wide variety of extracellular proteins.

[0052] Once bound to tissues, zymogen FVII is rapidly converted to FVIIa by limited proteolysis to form the active tissue factor-FVIIa complex. FVIIa, which circulates as an enzyme at a concentration of approximately 0.1 nM (1% of plasma FVII), can also bind directly to tissue factor. The allosteric interaction between tissue factor and FVIIa on the tissue factor-FVIIa complex greatly increases the enzymatic activity of FVIIa: approximately a 20- to 100-fold increase in the rate of hydrolysis of small chromogenic peptidyl substrates and an almost million-fold increase in the rate of activation of the natural polymer substrates FIX and FX. In response to the allosteric activation of the FVIIa active site upon binding to tissue factor, the tissue factor-FVIIa complex on the phospholipid bilayer (i.e., upon exposure of phosphatidyl-L-serine on the membrane surface) undergoes Ca activation. 2+The approximately one million-fold overall increase in FX activation by tissue factor-FVIIa-phospholipid complexes compared to free FVIIa represents a key control point in the coagulation cascade.

[0053] FVII is a single polypeptide chain of approximately 50 kDa consisting of 406 amino acid residues, which contains an N-terminal γ-carboxyglutamic acid-rich (GLA) domain, two epidermal growth factor-like domains (EGF1 and EGF2), and a C-terminal serine protease domain. FVII is a single polypeptide chain consisting of Ile- in the short linker region between the EGF2 and protease domains. 154 -Arg 152 It is activated to FVIIa by specific proteolytic cleavage of the Cys bond. 135 and Cys 262 FVIIa binds to Ca via its N-terminal GLA domain. 2+ Immediately C-terminal to the GLA domain is an aromatic stack and two EGF domains. The aromatic stack binds to a single Ca 2+ GLA connects to the EGF1 domain, which binds to Ca. 2+ Occupation of the binding site increases FVIIa amidolytic activity and tissue factor association. The catalytic triad consists of His 193 , Asp 242 , and Ser 344 and a single Ca domain in the FVIIa protease domain. 2+ Ion binding is important for its catalytic activity. Proteolytic activation of FVII to FVIIa occurs via Ile 153 releases the newly formed amino terminus, folds back, and inserts into the activation pocket, where it is Asp 343This salt bridge forms an oxyanion hole with the carboxylate of FVIIa. This salt bridge formation is important for FVIIa activity. However, in free FVIIa, oxyanion hole formation does not occur upon proteolytic activation. As a result, FVIIa circulates in a zymogen-like state that is poorly recognized by plasma protease inhibitors, allowing it to circulate with a half-life of approximately 90 minutes.

[0054] Tissue factor-mediated positioning of the FVIIa active site on the membrane surface is important for FVIIa to interact with its cognate substrate. Free FVIIa adopts a stable extended conformation when bound to a membrane, with its active site located approximately 80 Å above the membrane surface. Upon binding of FVIIa to tissue factor, the FVIIa active site relocates approximately 6 Å closer to the membrane. This adjustment may aid in the proper alignment of the FVIIa catalytic triad with the target substrate cleavage site. Using FVIIa lacking the GLA domain, it was shown that the active site was still located at a similar distance on the membrane, demonstrating that tissue factor can fully support the positioning of the FVIIa active site even in the absence of FVIIa-membrane interactions. Further data showed that tissue factor supported full FVIIa proteolytic activity as long as the tissue factor extracellular domain was somehow tethered to the membrane surface. However, elevating the active site of FVIIa above 80 Å of the membrane surface significantly reduced the ability of the tissue factor-FVIIa complex to activate FX, but did not reduce tissue factor-FVIIa amidolytic activity.

[0055] Alanine-scanning mutagenesis has been used to evaluate the role of specific amino acid side chains in the tissue factor extracellular domain for interaction with FVIIa (Gibbs et al., Biochemistry 33(47):14003-14010, 1994; Schullek et al., J Biol Chem 269(30):19399-19403, 1994). Alanine substitutions identified a limited number of residue positions where alanine substitutions caused a 5- to 10-fold lower affinity for FVIIa binding. Most of these residue side chains were found to be well exposed to solvent in the crystal structure, consistent with a macromolecular ligand interaction. The FVIIa ligand-binding site is located across an extensive region of the interface between the two modules. In the C module, residues Arg, located on the protruding BC loop, 135 and Phe 140 provides independent contact with FVIIa. 133 is located at the base of the finger-like structure and is packed into the gap between the two modules. 20 , Thr 60 , Asp 58 , and Ile 22 provides continuity to the main cluster of important binding residues consisting of Thr 60 is only partially solvent exposed and may play a local structural role rather than making significant contact with the ligand. 24 and Gln 110 , as well as potentially more distant residues Val 207 The binding region extends from Asp58 to Lys 48 , Lys 46 , Gln 37 , Asp 44 , and Trp 45 It extends over the convex surface area formed by Trp 45 and Asp 44 does not interact with FVIIa independently, which suggests that Trp 45 The effect of mutations at positions 44 and Gln 37This may reflect the structural importance of this side chain for local packing of the side chains. The interaction region further includes two surface-exposed aromatic residues, Phe 76 and Tyr 78 which form part of the hydrophobic cluster in the N module.

[0056] The known physiological substrates of tissue factor-FVIIa are FVII, FIX, and FX, as well as certain proteinase-activated receptors. Mutational analysis has identified a number of residues that, when mutated, support full FVIIa amidolytic activity toward small peptidyl substrates but lose their ability to support activation of macromolecular substrates (i.e., FVII, FIX, and FX) (Ruf et al., J Biol Chem 267(31):22206-22210, 1992; Ruf et al., J Biol Chem 267(9):6375-6381, 1992; Huang et al., J Biol Chem 271(36):21752-21757, 1996; Kirchhofer et al., Biochemistry 39(25):7380-7387, 2000). The tissue factor loop region of residues 159-165, and residues within or adjacent to this flexible loop, have been shown to be important for the proteolytic activity of the tissue factor-FVIIa complex. This defines a proposed substrate-binding exosite region of tissue factor that is far removed from the FVIIa active site. Substitution of glycine residues with the slightly bulky residue alanine significantly impairs tissue factor-FVIIa proteolytic activity. This suggests that the flexibility conferred by glycine is important for the loop of residues 159-165 for tissue factor macromolecular substrate recognition.

[0057] Residue Lys 165 and Lys 166 Lys has also been demonstrated to be important for substrate recognition and binding. Mutation of either of these residues to alanine results in a significant decrease in tissue factor cofactor function. 165 and Lys 166 are opposite to each other, and Lys 165points towards FVIIa in most tissue factor-FVIIa structures, and Lys 166 points towards the substrate-binding exosite region in the crystal structure. 165 and Gla 35 The putative salt bridge formation between tissue factor and the GLA domain of FVIIa supports the idea that tissue factor interaction with the GLA domain of FVIIa regulates substrate recognition. These results suggest that the C-terminal part of the tissue factor ectodomain interacts directly with the GLA domain of FIX and FX, and possibly the adjacent EGF1 domain, and that the presence of the FVIIa GLA domain may directly or indirectly regulate these interactions.

[0058] Soluble tissue factor domain In some embodiments of any of the polypeptides, compositions, or methods described herein, the soluble tissue factor domain can be a wild-type tissue factor polypeptide lacking the signal sequence, transmembrane domain, and intracellular domain. In some examples, the soluble tissue factor domain can be a tissue factor variant in which the wild-type tissue factor polypeptide lacks the signal sequence, transmembrane domain, and intracellular domain and is further modified in selected amino acids. In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble rat tissue factor domain. Non-limiting examples of soluble human tissue factor domains, mouse soluble tissue factor domains, rat soluble tissue factor domains, and mutant soluble tissue factor domains are provided below.

[0059] Exemplary soluble human tissue factor domain (SEQ ID NO: 1) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0060] Exemplary nucleic acid encoding a soluble human tissue factor domain (SEQ ID NO: 9) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACAC CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGAC AGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGC AAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

[0061] Exemplary mutant soluble human tissue factor domain (SEQ ID NO: 10) SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0062] Exemplary mutant soluble human tissue factor domain (SEQ ID NO: 11) SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDAKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLAENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0063] Exemplary soluble mouse tissue factor domain (SEQ ID NO: 12) agipekafnltwistdfktilewqpkptnytytvqisdrsrnwknkcfsttdtecdltdeivkdvtwayeakvlsvprrnsvhgdgdqlvihgeeppftnap kflpyrdtnlgqpviqqfeqdgrklnvvvkdsltlvrkngtfltlrqvfgkdlgyiityrkgsstgkktnitntnefsidveegvsycffvqamifsrktnqnspgsstvcteqwksflge

[0064] Exemplary soluble rat tissue factor domain (SEQ ID NO: 13) agtppgkafnltwistdfktilewqpkptnytytvqisdrsrnwkykctgttdtecdltdeivkdvnwtyearvlsvpwrnsthgketlfgthgeeppftnarkflpyrdtk igqpviqkyeqggtklkvtvkdsftlvrkngtfltlrqvfgndlgyiltyrkdsstgrktntthtneflidvekgvsycffaqavifsrktnhkspesitkcteqwksvlge

[0065] In some embodiments, the soluble tissue factor domain can comprise a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:1, 10, 11, 12, or 13. In some embodiments, the soluble tissue factor domain can comprise the sequence of SEQ ID NO: 1, 10, 11, 12, or 13 with 1 to 20 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) removed from its N-terminus and / or 1 to 20 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) removed from its C-terminus.

[0066] As can be understood in the art, one of skill in the art will understand that mutations in amino acids that are conserved among different mammalian species are more likely to reduce the activity and / or structural stability of a protein, while mutations in amino acids that are not conserved among different mammalian species are less likely to reduce the activity and / or structural stability of a protein.

[0067] In some examples of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain cannot bind to factor VIIa. In some examples of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain does not convert inactive factor X to factor Xa. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the multi-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

[0068] In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble rat tissue factor domain.

[0069] In some examples, the soluble tissue factor domain does not include one or more (2, 3, 4, 5, 6, or 7) of: a lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. In some embodiments, the mutant soluble tissue factor has the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11.

[0070] In some examples, the soluble tissue factor domain can be encoded by a nucleic acid comprising a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:9.

[0071] In some embodiments, the soluble tissue factor domain is from about 20 amino acids to about 220 amino acids, from about 20 amino acids to about 215 amino acids, from about 20 amino acids to about 210 amino acids, from about 20 amino acids to about 205 amino acids, from about 20 amino acids to about 200 amino acids, from about 20 amino acids to about 195 amino acids, from about 20 amino acids to about 190 amino acids, from about 20 amino acids to about 185 amino acids, from about 20 amino acids to about 180 amino acids, from about 20 amino acids to about 175 amino acids, from about 20 amino acids to about 170 amino acids, from about 20 amino acids to about 165 amino acids, from about 20 amino acids to about 16 ...85 amino acids, from about 20 amino acids to about 180 amino acids, from about 20 amino acids to about 185 amino acids, from about 20 amino acids to about 180 amino acids, from about 2 amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, 0 to about 80 amino acids, about 20 to about 75 amino acids, about 20 to about 70 amino acids, about 20 to about 60 amino acids, about 20 to about 50 amino acids, about 20 to about 40 amino acids, about 20 to about 30 amino acids, about 30 to about 220 amino acids, about 30 to about 215 amino acids, about 30 to about 210 amino acids, about 30 to about 205 amino acids, about 30 to about 200 amino acids, about 30 to about 195 amino acids, about 30 to about 190 amino acids, about 30 to about 185 amino acids, about 30 amino acids to about 180 amino acids, about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 155 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino acids, about 30 amino acids to about 110 amino acids,about 30 to about 105 amino acids, about 30 to about 100 amino acids, about 30 to about 95 amino acids, about 30 to about 90 amino acids, about 30 to about 85 amino acids, about 30 to about 80 amino acids, about 30 to about 75 amino acids, about 30 to about 70 amino acids, about 30 to about 60 amino acids, about 30 to about 50 amino acids, about 30 to about 40 amino acids, about 40 to about 220 amino acids, about 40 to about 215 amino acids, about 40 to about 210 amino acids, about 40 to about 205 amino acids, About 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino acids, about 40 amino acids to about 190 amino acids, about 40 amino acids to about 185 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 50 amino acids to about about 220 amino acids, about 50 to about 215 amino acids, about 50 to about 210 amino acids, about 50 to about 205 amino acids, about 50 to about 200 amino acids, about 50 to about 195 amino acids, about 50 to about 190 amino acids, about 50 to about 185 amino acids, about 50 to about 180 amino acids, about 50 to about 175 amino acids, about 50 to about 170 amino acids, about 50 to about 165 amino acids, about 50 to about 160 amino acids, about 50 to about 155 amino acids, about 50 to about 150 amino acids,About 50 to about 145 amino acids, about 50 to about 140 amino acids, about 50 to about 135 amino acids, about 50 to about 130 amino acids, about 50 to about 125 amino acids, about 50 to about 120 amino acids, about 50 to about 115 amino acids, about 50 to about 110 amino acids, about 50 to about 105 amino acids, about 50 to about 100 amino acids, about 50 to about 95 amino acids, about 50 to about 90 amino acids, about 50 to about 85 amino acids, about 50 to about 80 amino acids, about 50 to about 75 amino acids amino acids, about 50 to about 70 amino acids, about 50 to about 60 amino acids, about 60 to about 220 amino acids, about 60 to about 215 amino acids, about 60 to about 210 amino acids, about 60 to about 205 amino acids, about 60 to about 200 amino acids, about 60 to about 195 amino acids, about 60 to about 190 amino acids, about 60 to about 185 amino acids, about 60 to about 180 amino acids, about 60 to about 175 amino acids, about 60 to about 170 amino acids, about 60 to about 165 amino acids, about 60 to about 160 amino acids, about 60 amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 115 amino acids, about 60 amino acids to about 110 amino acids, about 60 amino acids to about 105 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 Amino acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to about 220 amino acids, about 70 amino acids to about 215 amino acids, about 70 amino acids to about 210 amino acids, about 70 amino acids to about 205 amino acids, about 70 amino acids to about 200 amino acids, about 70 amino acids to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids,About 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 Amino acids, about 70 to about 90 amino acids, about 70 to about 85 amino acids, about 70 to about 80 amino acids, about 80 to about 220 amino acids, about 80 to about 215 amino acids, about 80 to about 210 amino acids, about 80 to about 205 amino acids, about 80 to about 200 amino acids, about 80 to about 195 amino acids, about 80 to about 190 amino acids, about 80 to about 185 amino acids, about 80 to about 180 amino acids, about 80 to about 175 amino acids, about 80 to about 170 amino acids, about 80 to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids amino acids to about 90 amino acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 215 amino acids, about 90 amino acids to about 210 amino acids, about 90 amino acids to about 205 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids,About 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 215 amino acids, about 100 amino acids to about 210 amino acids, about 100 amino acids about 205 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 14 ... amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 215 amino acids, about 110 amino acids to about 210 amino acids, about 110 amino acids to about 205 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, 10 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110 amino acids to about 160 amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids,About 115 amino acids to about 220 amino acids, about 115 amino acids to about 215 amino acids, about 115 amino acids to about 210 amino acids, about 115 amino acids to about 205 amino acids, about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115, Amino acids up to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 220 amino acids amino acids, about 120 to about 215 amino acids, about 120 to about 210 amino acids, about 120 to about 205 amino acids, about 120 to about 200 amino acids, about 120 to about 195 amino acids, about 120 to about 190 amino acids, about 120 to about 185 amino acids, about 120 to about 180 amino acids, about 120 to about 175 amino acids, about 120 to about 170 amino acids, about 120 to about 165 amino acids, about 120 to about 160 amino acids, about 120 to about 155 amino acids, about 120 to about 1 50 amino acids, about 120 amino acids to about 145 amino acids, about 120 amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids, about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125 amino acids, about 125 amino acids to about 220 amino acids, about 125 amino acids to about 215 amino acids, about 125 amino acids to about 210 amino acids, about 125 amino acids to about 205 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to about 185 amino acids, about 125 amino acids amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids, about 125 amino acids to about 150 amino acids, about 125 amino acids to about 145 amino acids, about 125 amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids, about 125 amino acids to about 130 amino acids, about 130 amino acids to about 220 amino acids, about 130 amino acids to about 215 amino acids, about 130 amino acids to about 210 amino acids,About 130 amino acids to about 205 amino acids, about 130 amino acids to about 200 amino acids, about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 185 amino acids, about 130 amino acids to about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 Amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 220 amino acids, about 135 amino acids to about 215 amino acids, about 135 amino acids to about 210 amino acids, about 135 amino acids to about 205 amino acids, about 135 amino acids to about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 170 amino acids, about 135 amino acids to about 165 ...65 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 1 about 160 amino acids, about 135 amino acids to about 155 amino acids, about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145 amino acids, about 135 amino acids to about 140 amino acids, about 140 amino acids to about 220 amino acids, about 140 amino acids to about 215 amino acids, about 140 amino acids to about 210 amino acids, about 140 amino acids to about 205 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140 amino acids to about 180 amino acids, about 140 Amino acids to about 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to about 155 amino acids, about 140 amino acids to about 150 amino acids, about 140 amino acids to about 145 amino acids, about 145 amino acids to about 220 amino acids, about 145 amino acids to about 215 amino acids, about 145 amino acids to about 210 amino acids, about 145 amino acids to about 205 amino acids, about 145 amino acids to about 200 amino acids, about 145 amino acids to about 195 amino acids, about 145 amino acids to about 190 amino acids,About 145 amino acids to about 185 amino acids, about 145 amino acids to about 180 amino acids, about 145 amino acids to about 175 amino acids, about 145 amino acids to about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145 amino acids to about 160 amino acids, about 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 215 amino acids, about 150 amino acids to about 210 amino acids, about 150 amino acids to about 205 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 Amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to about 220 amino acids, about 155 amino acids to about 215 amino acids, about 155 amino acids to about 210 amino acids, about 155 amino acids to about 205 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 215 amino acids about 195 amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160 amino acids, about 160 amino acids to about 220 amino acids, about 160 amino acids to about 215 amino acids, about 160 amino acids to about 210 amino acids, about 160 amino acids to about 205 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195 amino acids, about 160 Amino acids to about 190 amino acids, about 160 amino acids to about 185 amino acids, about 160 amino acids to about 180 amino acids, about 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids, about 165 amino acids to about 220 amino acids, about 165 amino acids to about 215 amino acids, about 165 amino acids to about 210 amino acids, about 165 amino acids to about 205 amino acids, about 165 amino acids to about 200 amino acids, about 165 amino acids to about 195 amino acids, about 165 amino acids to about 190 amino acids, about 165 amino acids to about 185 amino acids,About 165 amino acids to about 180 amino acids, about 165 amino acids to about 175 amino acids, about 165 amino acids to about 170 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 215 amino acids, about 170 amino acids to about 210 amino acids, about 170 amino acids to about 205 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 220 Amino acids, about 175 amino acids to about 215 amino acids, about 175 amino acids to about 210 amino acids, about 175 amino acids to about 205 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 195 amino acids, about 175 amino acids to about 190 amino acids, about 175 amino acids to about 185 amino acids, about 175 amino acids to about 180 amino acids, about 180 amino acids to about 220 amino acids, about 180 amino acids to about 215 amino acids, about 180 amino acids to about 210 amino acids, about 180 amino acids to about 205 amino acids, about 180 amino acids to about 20 ... about 195 amino acids, about 180 amino acids to about 190 amino acids, about 180 amino acids to about 185 amino acids, about 185 amino acids to about 220 amino acids, about 185 amino acids to about 215 amino acids, about 185 amino acids to about 210 amino acids, about 185 amino acids to about 205 amino acids, about 185 amino acids to about 200 amino acids, about 185 amino acids to about 195 amino acids, about 185 amino acids to about 190 amino acids, about 190 amino acids to about 220 amino acids, about 190 amino acids to about 215 amino acids, about 190 amino acids to about 210 amino acids, about 190 amino acids to about 205 amino acids, about 190 Amino acids to about 200 amino acids, about 190 amino acids to about 195 amino acids, about 195 amino acids to about 220 amino acids, about 195 amino acids to about 215 amino acids, about 195 amino acids to about 210 amino acids, about 195 amino acids to about 205 amino acids, about 195 amino acids to about 200 amino acids, about 200 amino acids to about 220 amino acids, about 200 amino acids to about 215 amino acids, about 200 amino acids to about 210 amino acids, about 200 amino acids to about 205 amino acids, about 205 amino acids to about 220 amino acids, about 205 amino acids to about 215 amino acids, about 205 amino acids to about 210 amino acids,It can have a total length of about 210 to about 220 amino acids, about 210 to about 215 amino acids, or about 215 to about 220 amino acids.

[0072] Linker sequence In some embodiments, the linker sequence can be a flexible linker sequence. Non-limiting examples of linker sequences that can be used are described in Klein et al., Protein Engineering, Design & Selection 27(10):325-330, 2014; Priyanka et al., Protein Sci. 22(2):153-167, 2013. In some examples, the linker sequence is a synthetic linker sequence.

[0073] In some embodiments of any of the multi-chain chimeric polypeptides described herein, a first chimeric polypeptide can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art). In some embodiments of any of the multi-chain chimeric polypeptides described herein, a second chimeric polypeptide can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art).

[0074] In some embodiments, the linker sequence may be from 1 to about 100 amino acids, from 1 to about 90 amino acids, from 1 to about 80 amino acids, from 1 to about 70 amino acids, from 1 to about 60 amino acids, from 1 to about 50 amino acids, from 1 to about 45 amino acids, from 1 to about 40 amino acids, from 1 to about 35 amino acids, from 1 to about 30 amino acids, from 1 to about 25 amino acids, from 1 to about 24 amino acids, from 1 to about 22 amino acids, from 1 to about 20 amino acids, from 1 to about 18 amino acids, from 1 to about 16 amino acids, or from 1 to about 2 to about 14 amino acids, 1 to about 12 amino acids, 1 to about 10 amino acids, 1 to about 8 amino acids, 1 to about 6 amino acids, 1 to about 4 amino acids, about 2 to about 100 amino acids, about 2 to about 90 amino acids, about 2 to about 80 amino acids, about 2 to about 70 amino acids, about 2 to about 60 amino acids, about 2 to about 50 amino acids, about 2 to about 45 amino acids, about 2 to about 40 amino acids, about 2 to about 35 amino acids, about 2 to about 30 amino acids, about 2 to about 25 amino acids, about 2 to about 3 About 24 amino acids, about 2 to about 22 amino acids, about 2 to about 20 amino acids, about 2 to about 18 amino acids, about 2 to about 16 amino acids, about 2 to about 14 amino acids, about 2 to about 12 amino acids, about 2 to about 10 amino acids, about 2 to about 8 amino acids, about 2 to about 6 amino acids, about 2 to about 4 amino acids, about 4 to about 100 amino acids, about 4 to about 90 amino acids, about 4 to about 80 amino acids, about 4 to about 70 amino acids, about 4 to about 60 amino acids, about 4 to about 50 amino acids, about 4 amino acids about 4 to about 45 amino acids, about 4 to about 40 amino acids, about 4 to about 35 amino acids, about 4 to about 30 amino acids, about 4 to about 25 amino acids, about 4 to about 24 amino acids, about 4 to about 22 amino acids, about 4 to about 20 amino acids, about 4 to about 18 amino acids, about 4 to about 16 amino acids, about 4 to about 14 amino acids, about 4 to about 12 amino acids, about 4 to about 10 amino acids, about 4 to about 8 amino acids, about 4 to about 6 amino acids, about 6 to about 100 amino acids, about 6 to about 90 amino acids,About 6 amino acids to about 80 amino acids, about 6 amino acids to about 70 amino acids, about 6 amino acids to about 60 amino acids, about 6 amino acids to about 50 amino acids, about 6 amino acids to about 45 amino acids, about 6 amino acids to about 40 amino acids, about 6 amino acids to about 35 amino acids, about 6 amino acids to about 30 amino acids, about 6 amino acids to about 25 amino acids, about 6 amino acids to about 24 amino acids, about 6 amino acids to about 22 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 18 amino acids, about 6 amino acids to about 16 amino acids, about 6 amino acids to about 14 amino acids, about 6 amino acids to about 12 amino acids, about 6 amino acids to about 1 About 10 amino acids, about 6 to about 8 amino acids, about 8 to about 100 amino acids, about 8 to about 90 amino acids, about 8 to about 80 amino acids, about 8 to about 70 amino acids, about 8 to about 60 amino acids, about 8 to about 50 amino acids, about 8 to about 45 amino acids, about 8 to about 40 amino acids, about 8 to about 35 amino acids, about 8 to about 30 amino acids, about 8 to about 25 amino acids, about 8 to about 24 amino acids, about 8 to about 22 amino acids, about 8 to about 20 amino acids, about 8 to about 18 amino acids , about 8 amino acids to about 16 amino acids, about 8 amino acids to about 14 amino acids, about 8 amino acids to about 12 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 70 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about 45 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 24 amino acids, about 10 to about 22 amino acids, about 10 to about 20 amino acids, about 10 to about 18 amino acids, about 10 to about 16 amino acids, about 10 to about 14 amino acids, about 10 to about 12 amino acids, about 12 to about 100 amino acids, about 12 to about 90 amino acids, about 12 to about 80 amino acids, about 12 to about 70 amino acids, about 12 to about 60 amino acids, about 12 to about 50 amino acids, about 12 to about 45 amino acids, about 12 to about 40 amino acids, about 12 to about 35 amino acids,About 12 amino acids to about 30 amino acids, about 12 amino acids to about 25 amino acids, about 12 amino acids to about 24 amino acids, about 12 amino acids to about 22 amino acids, about 12 amino acids to about 20 amino acids, about 12 amino acids to about 18 amino acids, about 12 amino acids to about 16 amino acids, about 12 amino acids to about 14 amino acids, about 14 amino acids to about 100 amino acids, about 14 amino acids to about 90 amino acids, about 14 amino acids to about 80 amino acids, about 14 amino acids to about 70 amino acids, about 14 amino acids to about 60 amino acids, about 14 amino acids to about 50 amino acids, about 14 amino acids to about 45 amino acids, about 14 amino acids to about 5 about 40 amino acids, about 14 amino acids to about 35 amino acids, about 14 amino acids to about 30 amino acids, about 14 amino acids to about 25 amino acids, about 14 amino acids to about 24 amino acids, about 14 amino acids to about 22 amino acids, about 14 amino acids to about 20 amino acids, about 14 amino acids to about 18 amino acids, about 14 amino acids to about 16 amino acids, about 16 amino acids to about 100 amino acids, about 16 amino acids to about 90 amino acids, about 16 amino acids to about 80 amino acids, about 16 amino acids to about 70 amino acids, about 16 amino acids to about 60 amino acids, about 16 amino acids to about 50 amino acids, about 16 amino acids to about 45 amino acids, About 16 amino acids to about 40 amino acids, about 16 amino acids to about 35 amino acids, about 16 amino acids to about 30 amino acids, about 16 amino acids to about 25 amino acids, about 16 amino acids to about 24 amino acids, about 16 amino acids to about 22 amino acids, about 16 amino acids to about 20 amino acids, about 16 amino acids to about 18 amino acids, about 18 amino acids to about 100 amino acids, about 18 amino acids to about 90 amino acids, about 18 amino acids to about 80 amino acids, about 18 amino acids to about 70 amino acids, about 18 amino acids to about 60 amino acids, about 18 amino acids to about 50 amino acids, about 18 amino acids to about 45 amino acids, about 18 amino acids to about about 40 amino acids, about 18 amino acids to about 35 amino acids, about 18 amino acids to about 30 amino acids, about 18 amino acids to about 25 amino acids, about 18 amino acids to about 24 amino acids, about 18 amino acids to about 22 amino acids, about 18 amino acids to about 20 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 35 amino acids,About 20 to about 30 amino acids, about 20 to about 25 amino acids, about 20 to about 24 amino acids, about 20 to about 22 amino acids, about 22 to about 100 amino acids, about 22 to about 90 amino acids, about 22 to about 80 amino acids, about 22 to about 70 amino acids, about 22 to about 60 amino acids, about 22 to about 50 amino acids, about 22 to about 45 amino acids, about 22 to about 40 amino acids, about 22 to about 35 amino acids, about 22 to about 30 amino acids, about 22 to about 25 amino acids, about 22 to about 24 amino acids, about 25 to about 100 amino acids, about 25 to about 90 amino acids, about 25 to about 80 amino acids, about 25 to about 70 amino acids, about 25 to about 60 amino acids, about 25 to about 50 amino acids, about 25 to about 45 amino acids, about 25 to about 40 amino acids, about 25 to about 35 amino acids, about 25 to about 30 amino acids, about 30 to about 100 amino acids, about 30 to about 90 amino acids, about 30 to about 80 amino acids, about 30 to about 70 amino acids, about 30 to about 60 amino acids, 30 to about 50 amino acids, about 30 to about 45 amino acids, about 30 to about 40 amino acids, about 30 to about 35 amino acids, about 35 to about 100 amino acids, about 35 to about 90 amino acids, about 35 to about 80 amino acids, about 35 to about 70 amino acids, about 35 to about 60 amino acids, about 35 to about 50 amino acids, about 35 to about 45 amino acids, about 35 to about 40 amino acids, about 40 to about 100 amino acids, about 40 to about 90 amino acids, about 40 to about 80 amino acids, about 40 to about 70 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 80 amino acids, about 45 amino acids to about 70 amino acids, about 45 amino acids to about 60 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 100 amino acids,It can have a total length of about 60 to about 90 amino acids, about 60 to about 80 amino acids, about 60 to about 70 amino acids, about 70 to about 100 amino acids, about 70 to about 90 amino acids, about 70 to about 80 amino acids, about 80 to about 100 amino acids, about 80 to about 90 amino acids, or about 90 to about 100 amino acids.

[0075] In some embodiments, the linker is rich in glycine (Gly or G) residues. In some embodiments, the linker is rich in serine (Ser or S) residues. In some embodiments, the linker is rich in glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs. In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS) sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Ser-Gly (GGSG) sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences.

[0076] In some embodiments, the linker sequence may comprise or consist of GGGGSGGGGSGGGGS (SEQ ID NO: 3). In some aspects, the linker sequence may be encoded by a nucleic acid comprising or consisting of GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID NO: 14). In some embodiments, the linker sequence may comprise or consist of GGGSGGGS (SEQ ID NO: 15).

[0077] Target Binding Domain In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and / or the additional target binding domain(s) can be an antigen binding domain that specifically binds to a ligand of TGF-βRII (e.g., any of the exemplary antigen binding domains described herein or known in the art), or a soluble interleukin or cytokine receptor that specifically binds to a ligand of TGF-βRII (e.g., any of the exemplary soluble interleukin receptors or soluble cytokine receptors described herein).

[0078] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and / or the one or more additional target binding domains are each independently from about 5 amino acids to about 1000 amino acids, from about 5 amino acids to about 950 amino acids, from about 5 amino acids to about 900 amino acids, from about 5 amino acids to about 850 amino acids, from about 5 amino acids to about 800 amino acids, from about 5 amino acids to about 750 amino acids, from about 5 amino acids to about 700 amino acids, from about 5 amino acids to about 650 amino acids, from about 5 amino acids to about 600 amino acids, from about 5 amino acids to about 700 amino acids, from about 5 amino acids to about 850 amino acids, from about 5 amino acids to about 800 amino acids, about 5 to about 550 amino acids, about 5 to about 500 amino acids, about 5 to about 450 amino acids, about 5 to about 400 amino acids, about 5 to about 350 amino acids, about 5 to about 300 amino acids, about 5 to about 280 amino acids, about 5 to about 260 amino acids, about 5 to about 240 amino acids, about 5 to about 220 amino acids, about 5 to about 200 amino acids, about 5 to about 195 amino acids, about 5 to about 190 amino acids, about 5 to about 185 amino acids, about 5 to about 180 amino acids, about 5 to about 175 amino acids acid, about 5 amino acids to about 170 amino acids, about 5 amino acids to about 165 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 155 amino acids, about 5 amino acids to about 150 amino acids, about 5 amino acids to about 145 amino acids, about 5 amino acids to about 140 amino acids, about 5 amino acids to about 135 amino acids, about 5 amino acids to about 130 amino acids, about 5 amino acids to about 125 amino acids, about 5 amino acids to about 120 amino acids, about 5 amino acids to about 115 amino acids, about 5 amino acids to about 110 amino acids, about 5 amino acids to about 105 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids about 95 amino acids, about 5 amino acids to about 90 amino acids, about 5 amino acids to about 85 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 75 amino acids, about 5 amino acids to about 70 amino acids, about 5 amino acids to about 65 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 55 amino acids, about 5 amino acids to about 50 amino acids, about 5 amino acids to about 45 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 35 amino acids, about 5 amino acids to about 30 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids,About 5 amino acids to about 10 amino acids, about 10 amino acids to about 1000 amino acids, about 10 amino acids to about 950 amino acids, about 10 amino acids to about 900 amino acids, about 10 amino acids to about 850 amino acids, about 10 amino acids to about 800 amino acids, about 10 amino acids to about 750 amino acids, about 10 amino acids to about 700 amino acids, about 10 amino acids to about 650 amino acids, about 10 amino acids to about 600 amino acids, about 10 amino acids to about 550 amino acids, about 10 amino acids to about 500 amino acids, about 10 amino acids to about 450 amino acids, about 10 amino acids to about 400 amino acids, about 10 amino acids to about 350 amino acids, about 10 amino acids to about 300 amino acids, about 10 amino acids to about 280 amino acids, about 10 amino acids to about 260 amino acids, about 10 amino acids to about 240 amino acids, about 10 amino acids to about 220 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 195 amino acids, about 10 amino acids to about 190 amino acids, about 10 amino acids to about 185 amino acids, about 10 amino acids to about 180 amino acids, about 10 amino acids to about 175 amino acids, about 10 amino acids to about 170 amino acids, about 10 amino acids to about 165 amino acids, about 10 amino acids to about 160 amino acids, about 10 amino acids to about 155 amino acids, about 10 amino acids to about 150 amino acids, about 10 amino acids to about 145 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 135 amino acids, about 10 amino acids to about 130 amino acids, about 10 amino acids to about 125 amino acids, about 10 amino acids to about 120 amino acids, about 10 amino acids to about 115 amino acids, about 10 amino acids to about 110 amino acids, about 10 amino acids to about 105 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 95 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 85 Amino acids, about 10 to about 80 amino acids, about 10 to about 75 amino acids, about 10 to about 70 amino acids, about 10 to about 65 amino acids, about 10 to about 60 amino acids, about 10 to about 55 amino acids, about 10 to about 50 amino acids, about 10 to about 45 amino acids, about 10 to about 40 amino acids, about 10 to about 35 amino acids, about 10 to about 30 amino acids, about 10 to about 25 amino acids, about 10 to about 20 amino acids, about 10 to about 15 amino acids, about 15 to about 1000 amino acids,About 15 amino acids to about 950 amino acids, about 15 amino acids to about 900 amino acids, about 15 amino acids to about 850 amino acids, about 15 amino acids to about 800 amino acids, about 15 amino acids to about 750 amino acids, about 15 amino acids to about 700 amino acids, about 15 amino acids to about 650 amino acids, about 15 amino acids to about 600 amino acids, about 15 amino acids to about 550 amino acids, about 15 amino acids to about 500 amino acids, about 15 amino acids to about 450 amino acids, about 15 amino acids to about 400 amino acids, about 15 amino acids to about 350 amino acids, about 15 amino acids to about 300 amino acids, about 15 amino acids to about 280 amino acids, about 15 amino acids to about 260 amino acids, about 15 amino acids to about 240 amino acids, about 15 amino acids to about 220 amino acids, about 15 amino acids to about 200 amino acids, about 15 amino acids to about 195 amino acids, about 15 amino acids to about 190 amino acids, about 15 amino acids to about 185 amino acids, about 15 amino acids to about 180 amino acids, about 15 amino acids to about 175 amino acids, about 15 amino acids to about 170 amino acids, about 15 amino acids to about 165 amino acids, about 15 amino acids to about 160 amino acids, about 15 amino acids to about 155 amino acids, about 15 amino acids to about 150 amino acids, about 15 to about 145 amino acids, about 15 to about 140 amino acids, about 15 to about 135 amino acids, about 15 to about 130 amino acids, about 15 to about 125 amino acids, about 15 to about 120 amino acids, about 15 to about 115 amino acids, about 15 to about 110 amino acids, about 15 to about 105 amino acids, about 15 to about 100 amino acids, about 15 to about 95 amino acids, about 15 to about 90 amino acids, about 15 to about 85 amino acids, about 15 to about 80 amino acids, about 15 to about 75 amino acids amino acids, about 15 to about 70 amino acids, about 15 to about 65 amino acids, about 15 to about 60 amino acids, about 15 to about 55 amino acids, about 15 to about 50 amino acids, about 15 to about 45 amino acids, about 15 to about 40 amino acids, about 15 to about 35 amino acids, about 15 to about 30 amino acids, about 15 to about 25 amino acids, about 15 to about 20 amino acids, about 20 to about 1000 amino acids, about 20 to about 950 amino acids, about 20 to about 900 amino acids, about 20 to about 850 amino acids,About 20 amino acids to about 800 amino acids, about 20 amino acids to about 750 amino acids, about 20 amino acids to about 700 amino acids, about 20 amino acids to about 650 amino acids, about 20 amino acids to about 600 amino acids, about 20 amino acids to about 550 amino acids, about 20 amino acids to about 500 amino acids, about 20 amino acids to about 450 amino acids, about 20 amino acids to about 400 amino acids, about 20 amino acids to about 350 amino acids, about 20 amino acids to about 300 amino acids, about 20 amino acids to about 280 amino acids, about 20 amino acids to about 260 amino acids, about 20 amino acids to about 240 amino acids, about 20 amino acids to about 220 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 195 amino acids, about 20 amino acids to about 190 amino acids, about 20 amino acids to about 185 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 175 amino acids, about 20 amino acids to about 170 amino acids, about 20 amino acids to about 165 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 65 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 55 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino acids to about 30 amino acids, about 20 amino acids to about 25 amino acids, about 25 amino acids to about 1000 amino acids, about 25 amino acids to about 950 amino acids, about 25 amino acids to about 900 amino acids, about 25 amino acids to about 850 amino acids, about 25 amino acids to about 800 amino acids, about 25 amino acids to about 750 amino acids, about 25 amino acids to about 700 amino acids, about 25 amino acids to about 650 amino acids,About 25 amino acids to about 600 amino acids, about 25 amino acids to about 550 amino acids, about 25 amino acids to about 500 amino acids, about 25 amino acids to about 450 amino acids, about 25 amino acids to about 400 amino acids, about 25 amino acids to about 350 amino acids, about 25 amino acids to about 300 amino acids, about 25 amino acids to about 280 amino acids, about 25 amino acids to about 260 amino acids, about 25 amino acids to about 240 amino acids, about 25 amino acids to about 220 amino acids, about 25 amino acids to about 200 amino acids, about 25 amino acids to about 195 amino acids, about 25 amino acids to about 190 amino acids, about 25 amino acids to about 185 amino acids, about 25 amino acids to about 180 amino acids, about 25 amino acids to about 175 amino acids, about 25 amino acids to about 170 amino acids, about 25 amino acids to about 165 amino acids, about 25 amino acids to about 160 amino acids, about 25 amino acids to about 155 amino acids, about 25 amino acids to about 150 amino acids, about 25 amino acids to about 145 amino acids, about 25 amino acids to about 140 amino acids, about 25 amino acids to about 135 amino acids, about 25 amino acids to about 130 amino acids, about 25 amino acids to about 125 amino acids, about 25 amino acids to about 120 amino acids, about 25 amino acids to about 115 amino acids, about 2 5 amino acids to about 110 amino acids, about 25 amino acids to about 105 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 95 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 85 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 75 amino acids, about 25 amino acids to about 70 amino acids, about 25 amino acids to about 65 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 55 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 5 amino acids to about 35 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 1000 amino acids, about 30 amino acids to about 950 amino acids, about 30 amino acids to about 900 amino acids, about 30 amino acids to about 850 amino acids, about 30 amino acids to about 800 amino acids, about 30 amino acids to about 750 amino acids, about 30 amino acids to about 700 amino acids, about 30 amino acids to about 650 amino acids, about 30 amino acids to about 600 amino acids, about 30 amino acids to about 550 amino acids, about 30 amino acids to about 500 amino acids, about 30 amino acids to about 450 amino acids, about 30 amino acids to about 400 amino acids,About 30 amino acids to about 350 amino acids, about 30 amino acids to about 300 amino acids, about 30 amino acids to about 280 amino acids, about 30 amino acids to about 260 amino acids, about 30 amino acids to about 240 amino acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 185 amino acids, about 3, 0 amino acids to about 180 amino acids, about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 155 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino acids, about 30 amino acids to about 11 0 amino acids, about 30 to about 105 amino acids, about 30 to about 100 amino acids, about 30 to about 95 amino acids, about 30 to about 90 amino acids, about 30 to about 85 amino acids, about 30 to about 80 amino acids, about 30 to about 75 amino acids, about 30 to about 70 amino acids, about 30 to about 65 amino acids, about 30 to about 60 amino acids, about 30 to about 55 amino acids, about 30 to about 50 amino acids, about 30 to about 45 amino acids, about 30 to about 40 amino acids, about 30 to about 35 amino acids , about 35 amino acids to about 1000 amino acids, about 35 amino acids to about 950 amino acids, about 35 amino acids to about 900 amino acids, about 35 amino acids to about 850 amino acids, about 35 amino acids to about 800 amino acids, about 35 amino acids to about 750 amino acids, about 35 amino acids to about 700 amino acids, about 35 amino acids to about 650 amino acids, about 35 amino acids to about 600 amino acids, about 35 amino acids to about 550 amino acids, about 35 amino acids to about 500 amino acids, about 35 amino acids to about 450 amino acids, about 35 amino acids to about 400 amino acids, about 35 amino acids to about 350 amino acids, about 35 amino acids about 300 amino acids, about 35 amino acids to about 280 amino acids, about 35 amino acids to about 260 amino acids, about 35 amino acids to about 240 amino acids, about 35 amino acids to about 220 amino acids, about 35 amino acids to about 200 amino acids, about 35 amino acids to about 195 amino acids, about 35 amino acids to about 190 amino acids, about 35 amino acids to about 185 amino acids, about 35 amino acids to about 180 amino acids, about 35 amino acids to about 175 amino acids, about 35 amino acids to about 170 amino acids, about 35 amino acids to about 165 amino acids, about 35 amino acids to about 160 amino acids, about 35 amino acids to about 155 amino acids,About 35 amino acids to about 150 amino acids, about 35 amino acids to about 145 amino acids, about 35 amino acids to about 140 amino acids, about 35 amino acids to about 135 amino acids, about 35 amino acids to about 130 amino acids, about 35 amino acids to about 125 amino acids, about 35 amino acids to about 120 amino acids, about 35 amino acids to about 115 amino acids, about 35 amino acids to about 110 amino acids, about 35 amino acids to about 105 amino acids, about 35 amino acids to about 100 amino acids, about 35 amino acids to about 95 amino acids, about 35 amino acids to about 90 amino acids, about 35 amino acids to about 85 amino acids, about 35 amino acids to about 80 amino acids amino acids, about 35 amino acids to about 75 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 65 amino acids, about 35 amino acids to about 60 amino acids, about 35 amino acids to about 55 amino acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 1000 amino acids, about 40 amino acids to about 950 amino acids, about 40 amino acids to about 900 amino acids, about 40 amino acids to about 850 amino acids, about 40 amino acids to about 800 amino acids, about 40 amino acids to about 750 amino acids, about 40 amino acids to about 700 amino acids amino acids, about 40 amino acids to about 650 amino acids, about 40 amino acids to about 600 amino acids, about 40 amino acids to about 550 amino acids, about 40 amino acids to about 500 amino acids, about 40 amino acids to about 450 amino acids, about 40 amino acids to about 400 amino acids, about 40 amino acids to about 350 amino acids, about 40 amino acids to about 300 amino acids, about 40 amino acids to about 280 amino acids, about 40 amino acids to about 260 amino acids, about 40 amino acids to about 240 amino acids, about 40 amino acids to about 220 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino acids, about 40 amino acids to about 190 amino acids, about 40 amino acids to about 185 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids,About 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 65 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 55 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 Amino acids up to about 1000 amino acids, about 45 amino acids to about 950 amino acids, about 45 amino acids to about 900 amino acids, about 45 amino acids to about 850 amino acids, about 45 amino acids to about 800 amino acids, about 45 amino acids to about 750 amino acids, about 45 amino acids to about 700 amino acids, about 45 amino acids to about 650 amino acids, about 45 amino acids to about 600 amino acids, about 45 amino acids to about 550 amino acids, about 45 amino acids to about 500 amino acids, about 45 amino acids to about 450 amino acids, about 45 amino acids to about 400 amino acids, about 45 amino acids to about 350 amino acids, about 45 amino acids to about 30 0 amino acids, about 45 amino acids to about 280 amino acids, about 45 amino acids to about 260 amino acids, about 45 amino acids to about 240 amino acids, about 45 amino acids to about 220 amino acids, about 45 amino acids to about 200 amino acids, about 45 amino acids to about 195 amino acids, about 45 amino acids to about 190 amino acids, about 45 amino acids to about 185 amino acids, about 45 amino acids to about 180 amino acids, about 45 amino acids to about 175 amino acids, about 45 amino acids to about 170 amino acids, about 45 amino acids to about 165 amino acids, about 45 amino acids to about 160 amino acids, about 45 amino acids to about 155 amino acids, about 45 Amino acids up to about 150 amino acids, about 45 amino acids to about 145 amino acids, about 45 amino acids to about 140 amino acids, about 45 amino acids to about 135 amino acids, about 45 amino acids to about 130 amino acids, about 45 amino acids to about 125 amino acids, about 45 amino acids to about 120 amino acids, about 45 amino acids to about 115 amino acids, about 45 amino acids to about 110 amino acids, about 45 amino acids to about 105 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 95 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 85 amino acids, about 45 amino acids to about 80 amino acids,About 45 to about 75 amino acids, about 45 to about 70 amino acids, about 45 to about 65 amino acids, about 45 to about 60 amino acids, about 45 to about 55 amino acids, about 45 to about 50 amino acids, about 50 to about 1000 amino acids, about 50 to about 950 amino acids, about 50 to about 900 amino acids, about 50 to about 850 amino acids, about 50 to about 800 amino acids, about 50 to about 750 amino acids, about 50 to about 700 amino acids, about 50 to about 650 amino acids, about 50 to about 600 amino acids amino acids, about 50 to about 550 amino acids, about 50 to about 500 amino acids, about 50 to about 450 amino acids, about 50 to about 400 amino acids, about 50 to about 350 amino acids, about 50 to about 300 amino acids, about 50 to about 280 amino acids, about 50 to about 260 amino acids, about 50 to about 240 amino acids, about 50 to about 220 amino acids, about 50 to about 200 amino acids, about 50 to about 195 amino acids, about 50 to about 190 amino acids, about 50 to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 145 amino acids, about 50 amino acids to about 140 amino acids, about 50 amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino acids, about 50 amino acids to about 115 amino acids, about 50 amino acids to about 110 amino acids , about 50 amino acids to about 105 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 95 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 75 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 65 amino acids, about 50 amino acids to about 60 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 1000 amino acids, about 55 amino acids to about 950 amino acids, about 55 amino acids to about 900 amino acids, about 55 amino acids to about 850 amino acids,About 55 amino acids to about 800 amino acids, about 55 amino acids to about 750 amino acids, about 55 amino acids to about 700 amino acids, about 55 amino acids to about 650 amino acids, about 55 amino acids to about 600 amino acids, about 55 amino acids to about 550 amino acids, about 55 amino acids to about 500 amino acids, about 55 amino acids to about 450 amino acids, about 55 amino acids to about 400 amino acids, about 55 amino acids to about 350 amino acids, about 55 amino acids to about 300 amino acids, about 55 amino acids to about 280 amino acids, about 55 amino acids to about 260 amino acids, about 55 amino acids to about 240 amino acids, about 55 amino acids to about 22 0 amino acids, about 55 amino acids to about 200 amino acids, about 55 amino acids to about 195 amino acids, about 55 amino acids to about 190 amino acids, about 55 amino acids to about 185 amino acids, about 55 amino acids to about 180 amino acids, about 55 amino acids to about 175 amino acids, about 55 amino acids to about 170 amino acids, about 55 amino acids to about 165 amino acids, about 55 amino acids to about 160 amino acids, about 55 amino acids to about 155 amino acids, about 55 amino acids to about 150 amino acids, about 55 amino acids to about 145 amino acids, about 55 amino acids to about 140 amino acids, about 55 amino acids to about 135 amino acids, about 55 amino acids to about 130 amino acids, about 55 amino acids to about 125 amino acids, about 55 amino acids to about 120 amino acids, about 55 amino acids to about 115 amino acids, about 55 amino acids to about 110 amino acids, about 55 amino acids to about 105 amino acids, about 55 amino acids to about 100 amino acids, about 55 amino acids to about 95 amino acids, about 55 amino acids to about 90 amino acids, about 55 amino acids to about 85 amino acids, about 55 amino acids to about 80 amino acids, about 55 amino acids to about 75 amino acids, about 55 amino acids to about 70 amino acids, about 55 amino acids to about 65 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 1000 amino acids, about 60 amino acids to about 950 amino acids, about 60 amino acids to about 900 amino acids, about 60 amino acids to about 850 amino acids, about 60 amino acids to about 800 amino acids, about 60 amino acids to about 750 amino acids, about 60 amino acids to about 700 amino acids, about 60 amino acids to about 650 amino acids, about 60 amino acids to about 600 amino acids, about 60 amino acids to about 550 amino acids, about 60 amino acids to about 500 amino acids, about 60 amino acids to about 450 amino acids, about 60 amino acids to about 400 amino acids, about 60 amino acids to about 350 amino acids, about 60 amino acids to about 300 amino acids,About 60 amino acids to about 280 amino acids, about 60 amino acids to about 260 amino acids, about 60 amino acids to about 240 amino acids, about 60 amino acids to about 220 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 195 amino acids, about 60 amino acids to about 190 amino acids, about 60 amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 17, 5 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino acids to about 165 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 115 amino acids, about 60 amino acids to about 110 amino acids, about 60 amino acids to about 105 amino acids, about 6 0 amino acids to about 100 amino acids, about 60 amino acids to about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids, about 60 amino acids to about 65 amino acids, about 65 amino acids to about 1000 amino acids, about 65 amino acids to about 950 amino acids, about 65 amino acids to about 900 amino acids, about 65 amino acids to about 850 amino acids, about 65 amino acids to about 800 amino acids, about 65 amino acids to about 750 amino acids, about 65 amino acids to about 700 amino acids, About 65 amino acids to about 650 amino acids, about 65 amino acids to about 600 amino acids, about 65 amino acids to about 550 amino acids, about 65 amino acids to about 500 amino acids, about 65 amino acids to about 450 amino acids, about 65 amino acids to about 400 amino acids, about 65 amino acids to about 350 amino acids, about 65 amino acids to about 300 amino acids, about 65 amino acids to about 280 amino acids, about 65 amino acids to about 260 amino acids, about 65 amino acids to about 240 amino acids, about 65 amino acids to about 220 amino acids, about 65 amino acids to about 200 amino acids, about 65 amino acids to about 195 ... about 190 amino acids, about 65 amino acids to about 185 amino acids, about 65 amino acids to about 180 amino acids, about 65 amino acids to about 175 amino acids, about 65 amino acids to about 170 amino acids, about 65 amino acids to about 165 amino acids, about 65 amino acids to about 160 amino acids, about 65 amino acids to about 155 amino acids, about 65 amino acids to about 150 amino acids, about 65 amino acids to about 145 amino acids, about 65 amino acids to about 140 amino acids, about 65 amino acids to about 135 amino acids, about 65 amino acids to about 130 amino acids, about 65 amino acids to about 125 amino acids, about 65 amino acids to about 120 amino acids,About 65 amino acids to about 115 amino acids, about 65 amino acids to about 110 amino acids, about 65 amino acids to about 105 amino acids, about 65 amino acids to about 100 amino acids, about 65 amino acids to about 95 amino acids, about 65 amino acids to about 90 amino acids, about 65 amino acids to about 85 amino acids, about 65 amino acids to about 80 amino acids, about 65 amino acids to about 75 amino acids, about 65 amino acids to about 70 amino acids, about 70 amino acids to about 1000 amino acids, about 70 amino acids to about 950 amino acids, about 70 amino acids to about 900 amino acids, about 70 amino acids to about 850 amino acids, about 70 amino acids to about 800 amino acids amino acids, about 70 to about 750 amino acids, about 70 to about 700 amino acids, about 70 to about 650 amino acids, about 70 to about 600 amino acids, about 70 to about 550 amino acids, about 70 to about 500 amino acids, about 70 to about 450 amino acids, about 70 to about 400 amino acids, about 70 to about 350 amino acids, about 70 to about 300 amino acids, about 70 to about 280 amino acids, about 70 to about 260 amino acids, about 70 to about 240 amino acids, about 70 to about 220 amino acids, about 70 to about 200 amino acids, about 70 amino acids to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids, about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 7 0 amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 70 amino acids to about 75 amino acids, about 75 amino acids to about 1000 amino acids, about 75 amino acids to about 950 amino acids, about 75 amino acids to about 900 amino acids, about 75 amino acids to about 850 amino acids,About 75 amino acids to about 800 amino acids, about 75 amino acids to about 750 amino acids, about 75 amino acids to about 700 amino acids, about 75 amino acids to about 650 amino acids, about 75 amino acids to about 600 amino acids, about 75 amino acids to about 550 amino acids, about 75 amino acids to about 500 amino acids, about 75 amino acids to about 450 amino acids, about 75 amino acids to about 400 amino acids, about 75 amino acids to about 350 amino acids, about 75 amino acids to about 300 amino acids, about 75 amino acids to about 280 amino acids, about 75 amino acids to about 260 amino acids, about 75 amino acids to about 240 amino acids, about 75 amino acids to about 220 Amino acids, about 75 to about 200 amino acids, about 75 to about 195 amino acids, about 75 to about 190 amino acids, about 75 to about 185 amino acids, about 75 to about 180 amino acids, about 75 to about 175 amino acids, about 75 to about 170 amino acids, about 75 to about 165 amino acids, about 75 to about 160 amino acids, about 75 to about 155 amino acids, about 75 to about 150 amino acids, about 75 to about 145 amino acids, about 75 to about 140 amino acids, about 75 to about 135 amino acids, about 75 amino acids about 130 amino acids, about 75 amino acids to about 125 amino acids, about 75 amino acids to about 120 amino acids, about 75 amino acids to about 115 amino acids, about 75 amino acids to about 110 amino acids, about 75 amino acids to about 105 amino acids, about 75 amino acids to about 100 amino acids, about 75 amino acids to about 95 amino acids, about 75 amino acids to about 90 amino acids, about 75 amino acids to about 85 amino acids, about 75 amino acids to about 80 amino acids, about 80 amino acids to about 1000 amino acids, about 80 amino acids to about 950 amino acids, about 80 amino acids to about 900 amino acids, about 80 amino acids to about 850 amino acids, about 80 amino acids from about 800 amino acids, from about 80 amino acids to about 750 amino acids, from about 80 amino acids to about 700 amino acids, from about 80 amino acids to about 650 amino acids, from about 80 amino acids to about 600 amino acids, from about 80 amino acids to about 550 amino acids, from about 80 amino acids to about 500 amino acids, from about 80 amino acids to about 450 amino acids, from about 80 amino acids to about 400 amino acids, from about 80 amino acids to about 350 amino acids, from about 80 amino acids to about 300 amino acids, from about 80 amino acids to about 280 amino acids, from about 80 amino acids to about 260 amino acids, from about 80 amino acids to about 240 amino acids, from about 80 amino acids to about 220 amino acids,About 80 amino acids to about 200 amino acids, about 80 amino acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80 amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 175 amino acids, about 80 amino acids to about 170 amino acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 Amino acids, about 80 to about 125 amino acids, about 80 to about 120 amino acids, about 80 to about 115 amino acids, about 80 to about 110 amino acids, about 80 to about 105 amino acids, about 80 to about 100 amino acids, about 80 to about 95 amino acids, about 80 to about 90 amino acids, about 80 to about 85 amino acids, about 85 to about 1000 amino acids, about 85 to about 950 amino acids, about 85 to about 900 amino acids, about 85 to about 850 amino acids, about 85 to about 800 amino acids, about 85 to about 750 amino acids, about 85 amino acids to about 700 amino acids, about 85 amino acids to about 650 amino acids, about 85 amino acids to about 600 amino acids, about 85 amino acids to about 550 amino acids, about 85 amino acids to about 500 amino acids, about 85 amino acids to about 450 amino acids, about 85 amino acids to about 400 amino acids, about 85 amino acids to about 350 amino acids, about 85 amino acids to about 300 amino acids, about 85 amino acids to about 280 amino acids, about 85 amino acids to about 260 amino acids, about 85 amino acids to about 240 amino acids, about 85 amino acids to about 220 amino acids, about 85 amino acids to about 200 amino acids, about 85 amino acids amino acids to about 195 amino acids, about 85 amino acids to about 190 amino acids, about 85 amino acids to about 185 amino acids, about 85 amino acids to about 180 amino acids, about 85 amino acids to about 175 amino acids, about 85 amino acids to about 170 amino acids, about 85 amino acids to about 165 amino acids, about 85 amino acids to about 160 amino acids, about 85 amino acids to about 155 amino acids, about 85 amino acids to about 150 amino acids, about 85 amino acids to about 145 amino acids, about 85 amino acids to about 140 amino acids, about 85 amino acids to about 135 amino acids, about 85 amino acids to about 130 amino acids, about 85 amino acids to about 125 amino acids,About 85 amino acids to about 120 amino acids, about 85 amino acids to about 115 amino acids, about 85 amino acids to about 110 amino acids, about 85 amino acids to about 105 amino acids, about 85 amino acids to about 100 amino acids, about 85 amino acids to about 95 amino acids, about 85 amino acids to about 90 amino acids, about 90 amino acids to about 1000 amino acids, about 90 amino acids to about 950 amino acids, about 90 amino acids to about 900 amino acids, about 90 amino acids to about 850 amino acids, about 90 amino acids to about 800 amino acids, about 90 amino acids to about 750 amino acids, about 90 amino acids to about 700 amino acids, about 90 amino acids to about 650 amino acids, about 90 amino acids to about 600 amino acids, about 90 amino acids to about 550 amino acids, about 90 amino acids to about 500 amino acids, about 90 amino acids to about 450 amino acids, about 90 amino acids to about 400 amino acids, about 90 amino acids to about 350 amino acids, about 90 amino acids to about 300 amino acids, about 90 amino acids to about 280 amino acids, about 90 amino acids to about 260 amino acids, about 90 amino acids to about 240 amino acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, About 90 amino acids to about 185 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 12 ... about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 90 amino acids to about 95 amino acids, about 95 amino acids to about 1000 amino acids, about 95 amino acids to about 950 amino acids, about 95 amino acids to about 900 amino acids, about 95 amino acids to about 850 amino acids, about 95 amino acids to about 800 amino acids, about 95 amino acids to about 750 amino acids, about 95 amino acids to about 700 amino acids, about 95 amino acids to about 650 amino acids, about 95 amino acids to about 600 amino acids, about 95 amino acids to about 550 amino acids,About 95 amino acids to about 500 amino acids, about 95 amino acids to about 450 amino acids, about 95 amino acids to about 400 amino acids, about 95 amino acids to about 350 amino acids, about 95 amino acids to about 300 amino acids, about 95 amino acids to about 280 amino acids, about 95 amino acids to about 260 amino acids, about 95 amino acids to about 240 amino acids, about 95 amino acids to about 220 amino acids, about, 95 amino acids to about 200 amino acids, about 95 amino acids to about 195 amino acids, about 95 amino acids to about 190 amino acids, about 95 amino acids to about 185 amino acids, about 95 amino acids to about 180 amino acids, about 95 amino acids to about 175 amino acids, about 95 amino acids to about 170 amino acids, about 95 amino acids to about 165 amino acids, about 95 amino acids to about 160 amino acids, about 95 amino acids to about 155 amino acids, about 95 amino acids to about 150 amino acids, about 95 amino acids to about 145 amino acids, about 95 amino acids to about 140 amino acids, about 95 amino acids to about 135 amino acids, about 95 amino acids to about 130 amino acids, about 95 amino acids to about 125 amino acids, about 95 amino acids to about 120 amino acids, about 95 amino acids to about 115 amino acids, about 95 amino acids to about 110 amino acids, about 95 amino acids to about 105 amino acids, about 95 amino acids to about 100 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 950 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 to about 600 amino acids, about 100 to about 550 amino acids, about 100 to about 500 amino acids, about 100 to about 450 amino acids, about 100 to about 400 amino acids, about 100 to about 350 amino acids, about 100 to about 300 amino acids, about 100 to about 280 amino acids, about 100 to about 260 amino acids, about 100 to about 240 amino acids, about 100 to about 220 amino acids, about 100 to about 200 amino acids, about 100 to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids,About 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 100 amino acids to about 105 amino acids, about 105 amino acids to about 1000 amino acids, about 105 amino acids to about 950 amino acids, about 105 amino acids to about 900 amino acids, about 105 amino acids to about 850 amino acids, about 105 amino acids to about 800 amino acids, about 105 amino acids to about 750 amino acids, about 105 amino acids to about 700 amino acids, about 105 amino acids to about 650 amino acids, about 105 amino acids to about 600 amino acids, about 105 amino acids to about 550 Amino acids, about 105 amino acids to about 500 amino acids, about 105 amino acids to about 450 amino acids, about 105 amino acids to about 400 amino acids, about 105 amino acids to about 350 amino acids, about 105 amino acids to about 300 amino acids, about 105 amino acids to about 280 amino acids, about 105 amino acids to about 260 amino acids, about 105 amino acids to about 240 amino acids, about 105 amino acids to about 220 amino acids, about 105 amino acids to about 200 amino acids, about 105 amino acids to about 195 amino acids, about 105 amino acids to about 190 amino acids, about 105 amino acids to about 185 ... about 180 amino acids, about 105 amino acids to about 175 amino acids, about 105 amino acids to about 170 amino acids, about 105 amino acids to about 165 amino acids, about 105 amino acids to about 160 amino acids, about 105 amino acids to about 155 amino acids, about 105 amino acids to about 150 amino acids, about 105 amino acids to about 145 amino acids, about 105 amino acids to about 140 amino acids, about 105 amino acids to about 135 amino acids, about 105 amino acids to about 130 amino acids, about 105 amino acids to about 125 amino acids, about 105 amino acids to about 120 amino acids, about 105 amino acids to about 115 ... amino acids to about 110 amino acids, about 110 amino acids to about 1000 amino acids, about 110 amino acids to about 950 amino acids, about 110 amino acids to about 900 amino acids, about 110 amino acids to about 850 amino acids, about 110 amino acids to about 800 amino acids, about 110 amino acids to about 750 amino acids, about 110 amino acids to about 700 amino acids, about 110 amino acids to about 650 amino acids, about 110 amino acids to about 600 amino acids, about 110 amino acids to about 550 amino acids, about 110 amino acids to about 500 amino acids, about 110 amino acids to about 450 amino acids, about 110 amino acids to about 400 amino acids,About 110 amino acids to about 350 amino acids, about 110 amino acids to about 300 amino acids, about 110 amino acids to about 280 amino acids, about 110 amino acids to about 260 amino acids, about 110 amino acids to about 240 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 Amino acids, about 110 amino acids to about 160 amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to about 1000 amino acids, about 115 amino acids to about 950 amino acids, about 115 amino acids to about 900 amino acids, about 115 amino acids to about 850 amino acids, about 115 amino acids to about 800 amino acids, about 115 amino acids to about 750 amino acids, about 115 amino acids to about 700 amino acids, about 115 amino acids to about 650 amino acids, about 115 amino acids to about 600 amino acids, about 115 amino acids to about 550 amino acids, about 115 amino acids to about 500 amino acids, about 115 amino acids to about 450 amino acids, about 115 amino acids to about 400 amino acids, about 115 amino acids to about 350 amino acids, about 115 amino acids to about 300 amino acids, about 115 amino acids to about 280 amino acids, about 115 amino acids to about 260 amino acids, about 115 Amino acids to about 240 amino acids, about 115 amino acids to about 220 amino acids, about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids,About 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 1000 amino acids, about 120 amino acids to about 950 amino acids, about 120 amino acids to about 900 amino acids, about 120 amino acids to about 850 amino acids, about 120 amino acids to about 800 amino acids, about 120 amino acids to about 750 amino acids, about 120 amino acids to about 700 amino acids, about 120 amino acids to about 650 amino acids, about 120 amino acids to about 600 Amino acids, about 120 amino acids to about 550 amino acids, about 120 amino acids to about 500 amino acids, about 120 amino acids to about 450 amino acids, about 120 amino acids to about 400 amino acids, about 120 amino acids to about 350 amino acids, about 120 amino acids to about 300 amino acids, about 120 amino acids to about 280 amino acids, about 120 amino acids to about 260 amino acids, about 120 amino acids to about 240 amino acids, about 120 amino acids to about 220 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to about 2 About 185 amino acids, about 120 amino acids to about 180 amino acids, about 120 amino acids to about 175 amino acids, about 120 amino acids to about 170 amino acids, about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 155 amino acids, about 120 amino acids to about 150 amino acids, about 120 amino acids to about 145 amino acids, about 120 amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids, about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125 amino acids, about 125 amino acids to about 1000 amino acids, about 125 Amino acids to about 950 amino acids, about 125 amino acids to about 900 amino acids, about 125 amino acids to about 850 amino acids, about 125 amino acids to about 800 amino acids, about 125 amino acids to about 750 amino acids, about 125 amino acids to about 700 amino acids, about 125 amino acids to about 650 amino acids, about 125 amino acids to about 600 amino acids, about 125 amino acids to about 550 amino acids, about 125 amino acids to about 500 amino acids, about 125 amino acids to about 450 amino acids, about 125 amino acids to about 400 amino acids, about 125 amino acids to about 350 amino acids, about 125 amino acids to about 300 amino acids,About 125 amino acids to about 280 amino acids, about 125 amino acids to about 260 amino acids, about 125 amino acids to about 240 amino acids, about 125 amino acids to about 220 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids amino acids, about 125 amino acids to about 150 amino acids, about 125 amino acids to about 145 amino acids, about 125 amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids, about 125 amino acids to about 130 amino acids, about 130 amino acids to about 1000 amino acids, about 130 amino acids to about 950 amino acids, about 130 amino acids to about 900 amino acids, about 130 amino acids to about 850 amino acids, about 130 amino acids to about 800 amino acids, about 130 amino acids to about 750 amino acids, about 130 amino acids to about 700 amino acids, about 130 amino acids to about 650 amino acids, about 130 amino acids to about 1000 amino acids About 600 amino acids, about 130 amino acids to about 550 amino acids, about 130 amino acids to about 500 amino acids, about 130 amino acids to about 450 amino acids, about 130 amino acids to about 400 amino acids, about 130 amino acids to about 350 amino acids, about 130 amino acids to about 300 amino acids, about 130 amino acids to about 280 amino acids, about 130 amino acids to about 260 amino acids, about 130 amino acids to about 240 amino acids, about 130 amino acids to about 220 amino acids, about 130 amino acids to about 200 amino acids, about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids amino acids to about 185 amino acids, about 130 amino acids to about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 1000 amino acids, about 135 amino acids to about 950 amino acids, about 135 amino acids to about 900 amino acids,About 135 amino acids to about 850 amino acids, about 135 amino acids to about 800 amino acids, about 135 amino acids to about 750 amino acids, about 135 amino acids to about 700 amino acids, about 135 amino acids to about 650 amino acids, about 135 amino acids to about 600 amino acids, about 135 amino acids to about 550 amino acids, about 135 amino acids to about 500 amino acids, about 135 amino acids to about 450 amino acids, about 135, Amino acids up to about 400 amino acids, about 135 amino acids to about 350 amino acids, about 135 amino acids to about 300 amino acids, about 135 amino acids to about 280 amino acids, about 135 amino acids to about 260 amino acids, about 135 amino acids to about 240 amino acids, about 135 amino acids to about 220 amino acids, about 135 amino acids to about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 170 amino acids , about 135 amino acids to about 165 amino acids, about 135 amino acids to about 160 amino acids, about 135 amino acids to about 155 amino acids, about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145 amino acids, about 135 amino acids to about 140 amino acids, about 140 amino acids to about 1000 amino acids, about 140 amino acids to about 950 amino acids, about 140 amino acids to about 900 amino acids, about 140 amino acids to about 850 amino acids, about 140 amino acids to about 800 amino acids, about 140 amino acids to about 750 amino acids, about 140 amino acids to about 700 amino acids, about 140 amino acids to about 6 50 amino acids, about 140 amino acids to about 600 amino acids, about 140 amino acids to about 550 amino acids, about 140 amino acids to about 500 amino acids, about 140 amino acids to about 450 amino acids, about 140 amino acids to about 400 amino acids, about 140 amino acids to about 350 amino acids, about 140 amino acids to about 300 amino acids, about 140 amino acids to about 280 amino acids, about 140 amino acids to about 260 amino acids, about 140 amino acids to about 240 amino acids, about 140 amino acids to about 220 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to about 155 amino acids, about 140 amino acids to about 150 amino acids, about 140 amino acids to about 145 amino acids, about 145 amino acids to about 1000 amino acids, about 145 amino acids to about 950 amino acids, about 145 amino acids to about 900 amino acids, about 145 amino acids to about 850 amino acids,About 145 amino acids to about 800 amino acids, about 145 amino acids to about 750 amino acids, about 145 amino acids to about 700 amino acids, about 145 amino acids to about 650 amino acids, about 145 amino acids to about 600 amino acids, about 145 amino acids to about 550 amino acids, about 145 amino acids to about 500 amino acids, about 145 amino acids to about 450 amino acids, about 145 amino acids to about 400 amino acids, about 145 amino acids to about 350 amino acids, about 145 amino acids to about 300 amino acids, about 145 amino acids to about 280 amino acids, about 145 amino acids to about 260 amino acids, about 145 amino acids to about 240 Amino acids, about 145 to about 220 amino acids, about 145 to about 200 amino acids, about 145 to about 195 amino acids, about 145 to about 190 amino acids, about 145 to about 185 amino acids, about 145 to about 180 amino acids, about 145 to about 175 amino acids, about 145 to about 170 amino acids, about 145 to about 165 amino acids, about 145 to about 160 amino acids, about 145 to about 155 amino acids, about 145 to about 150 amino acids, about 150 to about 1000 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids to about 850 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 Amino acids to about 280 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids,About 155 amino acids to about 1000 amino acids, about 155 amino acids to about 950 amino acids, about 155 amino acids to about 900 amino acids, about 155 amino acids to about 850 amino acids, about 155 amino acids to about 800 amino acids, about 155 amino acids to about 750 amino acids, about 155 amino acids to about 700 amino acids, about 155 amino acids to about 650 amino acids, about 155 amino acids to about 600 amino acids, about 155 amino acids to about 550 amino acids, about 155 amino acids to about 500 amino acids, about 155 amino acids to about 450 amino acids, about 155 amino acids to about 400 amino acids, about 155 amino acids to about 350 Amino acids, about 155 amino acids to about 300 amino acids, about 155 amino acids to about 280 amino acids, about 155 amino acids to about 260 amino acids, about 155 amino acids to about 240 amino acids, about 155 amino acids to about 220 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 ...65 amino acids, about 155 amino acids to about 195 amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 1 About 160 amino acids, about 160 amino acids to about 1000 amino acids, about 160 amino acids to about 950 amino acids, about 160 amino acids to about 900 amino acids, about 160 amino acids to about 850 amino acids, about 160 amino acids to about 800 amino acids, about 160 amino acids to about 750 amino acids, about 160 amino acids to about 700 amino acids, about 160 amino acids to about 650 amino acids, about 160 amino acids to about 600 amino acids, about 160 amino acids to about 550 amino acids, about 160 amino acids to about 500 amino acids, about 160 amino acids to about 450 amino acids, about 160 amino acids to about 400 amino acids, about 160 Amino acids up to about 350 amino acids, about 160 amino acids to about 300 amino acids, about 160 amino acids to about 280 amino acids, about 160 amino acids to about 260 amino acids, about 160 amino acids to about 240 amino acids, about 160 amino acids to about 220 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195 amino acids, about 160 amino acids to about 190 amino acids, about 160 amino acids to about 185 amino acids, about 160 amino acids to about 180 amino acids, about 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids,About 165 amino acids to about 1000 amino acids, about 165 amino acids to about 950 amino acids, about 165 amino acids to about 900 amino acids, about 165 amino acids to about 850 amino acids, about 165 amino acids to about 800 amino acids, about 165 amino acids to about 750 amino acids, about 165 amino acids to about 700 amino acids, about 165 amino acids to about 650 amino acids, about 165 amino acids to about 600 amino acids, about 165 amino acids to about 550 amino acids, about 165 amino acids to about 500 amino acids, about 165 amino acids to about 450 amino acids, about 165 amino acids to about 400 amino acids, about 165 amino acids to about 350 Amino acids, about 165 amino acids to about 300 amino acids, about 165 amino acids to about 280 amino acids, about 165 amino acids to about 260 amino acids, about 165 amino acids to about 240 amino acids, about 165 amino acids to about 220 amino acids, about 165 amino acids to about 200 amino acids, about 165 amino acids to about 195 amino acids, about 165 amino acids to about 190 amino acids, about 165 amino acids to about 185 amino acids, about 165 amino acids to about 180 amino acids, about 165 amino acids to about 175 amino acids, about 165 amino acids to about 170 amino acids, about 170 amino acids to about 1000 amino acids, about 170 amino acids to about 950 amino acids, about 170 amino acids to about 900 amino acids, about 170 amino acids to about 850 amino acids, about 170 amino acids to about 800 amino acids, about 170 amino acids to about 750 amino acids, about 170 amino acids to about 700 amino acids, about 170 amino acids to about 650 amino acids, about 170 amino acids to about 600 amino acids, about 170 amino acids to about 550 amino acids, about 170 amino acids to about 500 amino acids, about 170 amino acids to about 450 amino acids, about 170 amino acids to about 400 amino acids, about 170 amino acids to about 350 amino acids, about 170 amino acids to about 300 amino acids, about 170 Amino acids to about 280 amino acids, about 170 amino acids to about 260 amino acids, about 170 amino acids to about 240 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 1000 amino acids, about 175 amino acids to about 950 amino acids, about 175 amino acids to about 900 amino acids, about 175 amino acids to about 850 amino acids,About 175 amino acids to about 800 amino acids, about 175 amino acids to about 750 amino acids, about 175 amino acids to about 700 amino acids, about 175 amino acids to about 650 amino acids, about 175 amino acids to about 600 amino acids, about 175 amino acids to about 550 amino acids, about 175 amino acids to about 500 amino acids, about 175 amino acids to about 450 amino acids, about 175 amino acids to about 400 amino acids, about 175 amino acids to about 350 amino acids, about 175 amino acids to about 300 amino acids, about 175 amino acids to about 280 amino acids, about 175 amino acids to about 260 amino acids, about 175 amino acids to about 240 amino acids amino acids, about 175 amino acids to about 220 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 195 amino acids, about 175 amino acids to about 190 amino acids, about 175 amino acids to about 185 amino acids, about 175 amino acids to about 180 amino acids, about 180 amino acids to about 1000 amino acids, about 180 amino acids to about 950 amino acids, about 180 amino acids to about 900 amino acids, about 180 amino acids to about 850 amino acids, about 180 amino acids to about 800 amino acids, about 180 amino acids to about 750 amino acids, about 180 amino acids to about 700 amino acids, about 180 amino acids to about 1000 amino acids About 650 amino acids, about 180 amino acids to about 600 amino acids, about 180 amino acids to about 550 amino acids, about 180 amino acids to about 500 amino acids, about 180 amino acids to about 450 amino acids, about 180 amino acids to about 400 amino acids, about 180 amino acids to about 350 amino acids, about 180 amino acids to about 300 amino acids, about 180 amino acids to about 280 amino acids, about 180 amino acids to about 260 amino acids, about 180 amino acids to about 240 amino acids, about 180 amino acids to about 220 amino acids, about 180 amino acids to about 200 amino acids, about 180 amino acids to about 195 amino acids, about 180 amino acids amino acids to about 190 amino acids, about 180 amino acids to about 185 amino acids, about 185 amino acids to about 1000 amino acids, about 185 amino acids to about 950 amino acids, about 185 amino acids to about 900 amino acids, about 185 amino acids to about 850 amino acids, about 185 amino acids to about 800 amino acids, about 185 amino acids to about 750 amino acids, about 185 amino acids to about 700 amino acids, about 185 amino acids to about 650 amino acids, about 185 amino acids to about 600 amino acids, about 185 amino acids to about 550 amino acids, about 185 amino acids to about 500 amino acids, about 185 amino acids to about 450 amino acids,About 185 amino acids to about 400 amino acids, about 185 amino acids to about 350 amino acids, about 185 amino acids to about 300 amino acids, about 185 amino acids to about 280 amino acids, about 185 amino acids to about 260 amino acids, about 185 amino acids to about 240 amino acids, about 185 amino acids to about 220 amino acids, about 185 amino acids to about 200 amino acids, about 185 amino acids to about 195 amino acids, about 1, 85 amino acids to about 190 amino acids, about 190 amino acids to about 1000 amino acids, about 190 amino acids to about 950 amino acids, about 190 amino acids to about 900 amino acids, about 190 amino acids to about 850 amino acids, about 190 amino acids to about 800 amino acids, about 190 amino acids to about 750 amino acids, about 190 amino acids to about 700 amino acids, about 190 amino acids to about 650 amino acids, about 190 amino acids to about 600 amino acids, about 190 amino acids to about 550 amino acids, about 190 amino acids to about 500 amino acids, about 190 amino acids to about 450 amino acids, about 190 amino acids to about 400 amino acids amino acids, about 190 amino acids to about 350 amino acids, about 190 amino acids to about 300 amino acids, about 190 amino acids to about 280 amino acids, about 190 amino acids to about 260 amino acids, about 190 amino acids to about 240 amino acids, about 190 amino acids to about 220 amino acids, about 190 amino acids to about 200 amino acids, about 190 amino acids to about 195 amino acids, about 195 amino acids to about 1000 amino acids, about 195 amino acids to about 950 amino acids, about 195 amino acids to about 900 amino acids, about 195 amino acids to about 850 amino acids, about 195 amino acids to about 800 amino acids, about 195 amino acids to about about 750 amino acids, about 195 amino acids to about 700 amino acids, about 195 amino acids to about 650 amino acids, about 195 amino acids to about 600 amino acids, about 195 amino acids to about 550 amino acids, about 195 amino acids to about 500 amino acids, about 195 amino acids to about 450 amino acids, about 195 amino acids to about 400 amino acids, about 195 amino acids to about 350 amino acids, about 195 amino acids to about 300 amino acids, about 195 amino acids to about 280 amino acids, about 195 amino acids to about 260 amino acids, about 195 amino acids to about 240 amino acids, about 195 amino acids to about 220 amino acids, about 195 amino acids amino acids to about 200 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids,About 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220 amino acids to about 900 amino acids, about 220 amino acids to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750 amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 Amino acids, about 220 amino acids to about 600 amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to about 500 amino acids, about 220 amino acids to about 450 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 350 amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to about 280 amino acids, about 220 amino acids to about 260 amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids, about 240 amino acids to about 500 amino acids, about 240 amino acids to about 450 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 350 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 Amino acids to about 1000 amino acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 450 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 350 amino acids,About 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to about 500 amino acids, about 280 amino acids to about 450 Amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 350 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about About 500 amino acids, about 300 to about 450 amino acids, about 300 to about 400 amino acids, about 300 to about 350 amino acids, about 350 to about 1000 amino acids, about 350 to about 950 amino acids, about 350 to about 900 amino acids, about 350 to about 850 amino acids, about 350 to about 800 amino acids, about 350 to about 750 amino acids, about 350 to about 700 amino acids, about 350 to about 650 amino acids, about 350 to about 600 amino acids, about 350 to about 550 amino acids, about 350 Amino acids to about 500 amino acids, about 350 amino acids to about 450 amino acids, about 350 amino acids to about 400 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids,About 400 amino acids to about 450 amino acids, about 450 amino acids to about 1000 amino acids, about 450 amino acids to about 950 amino acids, about 450 amino acids to about 900 amino acids, about 450 amino acids to about 850 amino acids, about 450 amino acids to about 800 amino acids, about 450 amino acids to about 750 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 550 amino acids, about 450 amino acids to about 500 amino acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 Amino acids, about 500 to about 900 amino acids, about 500 to about 850 amino acids, about 500 to about 800 amino acids, about 500 to about 750 amino acids, about 500 to about 700 amino acids, about 500 to about 650 amino acids, about 500 to about 600 amino acids, about 500 to about 550 amino acids, about 550 to about 1000 amino acids, about 550 to about 950 amino acids, about 550 to about 900 amino acids, about 550 to about 850 amino acids, about 550 to about 800 amino acids, about 550 to about About 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 Amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids,The total number of amino acids may be about 750 to about 850 amino acids, about 750 to about 800 amino acids, about 800 to about 1000 amino acids, about 800 to about 950 amino acids, about 800 to about 900 amino acids, about 800 to about 850 amino acids, about 850 to about 1000 amino acids, about 850 to about 950 amino acids, about 850 to about 900 amino acids, about 900 to about 1000 amino acids, about 900 to about 950 amino acids, or about 950 to about 1000 amino acids.

[0079] Any target binding domain described herein may be present in a concentration of 1×10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, 1 x 10 -10 Less than M, 1 x 10 -11 Less than M, 1 x 10 -12 Less than M or 1 x 10 -13 The dissociation equilibrium constant (K D In some embodiments, the antigen binding protein constructs provided herein can bind to a ligand of TGF-βRII at a concentration of about 1×10 -3 M ~ approx. 1×10 -5 M, about 1 x 10 -4 M ~ approx. 1×10 -6 M, about 1 x 10 -5 M ~ approx. 1×10 -7 M, about 1 x 10 -6 M ~ approx. 1×10 -8 M, about 1 x 10 -7 M ~ approx. 1×10 -9 M, about 1 x 10 -8 M ~ approx. 1×10 -10 M, or approximately 1 x 10 -9 M ~ approx. 1×10 -11 K of M (inclusive) D The antibody can bind to the identified antigen.

[0080] Any target binding domain described herein may bind to a ligand of TGF-βRII (e.g., TGF-β) with a K of about 1 pM to about 30 nM. Dcan be combined with (For example, about 1 pM to about 25 nM, about 1 pM to about 20 nM, about 1 pM to about 15 nM, about 1 pM to about 10 nM, about 1 pM to about 5 nM, about 1 pM to about 2 nM, about 1 pM to about 1 nM, about 1 pM to about 950 pM, about 1 pM to about 900 pM, about 1 pM to about 850 pM, about 1 pM to about 800 pM, about 1 pM to about 750 pM, about 1 pM to about 700 pM, about 1 pM to about 650 pM, about 1 pM to about 600 pM, about 1 pM to about 550 pM, about 1 pM to about 500 pM, about 1 pM to about 450 pM, about 1 pM to about 400 pM, about 1 pM to about 350 pM, about 1 pM to about 300 pM) , about 1 pM to about 250 pM, about 1 pM to about 200 pM, about 1 pM to about 150 pM, about 1 pM to about 100 pM, about 1 pM to about 90 pM, about 1pM to about 80pM, about 1pM to about 70pM, about 1pM to about 60pM, about 1pM to about 50pM, about 1pM to about 40pM, about 1pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 4 pM, about 1 pM to about 3 pM, about 1 pM to about 2pM, about 2pM to about 30nM, about 2pM to about 25nM, about 2pM to about 20nM, about 2pM to about 15nM, about 2pM to about 10nM, about 2p M ~ about 5nM, about 2pM - about 2nM, about 2pM - about 1nM, about 2pM - about 950pM, about 2pM - about 900pM, about 2pM - about 850p M, about 2 pM to about 800 pM, about 2 pM to about 750 pM, about 2 pM to about 700 pM, about 2 pM to about 650 pM, about 2 pM to about 600 pM, Approximately 2 pM to approximately 550 pM, approximately 2 pM to approximately 500 pM, approximately 2 pM to approximately 450 pM, approximately 2 pM to approximately 400 pM, approximately 2 pM to approximately 350 pM, approximately 2pM to about 300pM, about 2pM to about 250pM, about 2pM to about 200pM, about 2pM to about 150pM, about 2pM to about 100pM, about 2p M ~ about 90pM, about 2pM - about 80pM, about 2pM - about 70pM, about 2pM - about 60pM, about 2pM - about 50pM, about 2pM - about 40p M, about 2 pM to about 30 pM, about 2 pM to about 20 pM, about 2 pM to about 10 pM, about 2 pM to about 5 pM, about 2 pM to about 4 pM, about 2 pM to about 3 pM, about 5 pM to about 30 nM, about 5 pM to about 25 nM, about 5 pM to about 20 nM, about 5 pM to about 15 nM, about 5 pM to about 10 nM, about 5 pM ~about 5nM, about 5pM to about 2nM, about 5pM to about 1nM, about 5pM to about 950pM, about 5pM to about 900pM, about 5pM to about 850pM,about 5 pM to about 800 pM, about 5 pM to about 750 pM, about 5 pM to about 700 pM, about 5 pM to about 650 pM, about 5 pM to about 600 pM, about 5 pM to about 550 pM, about 5 pM to about 500 pM, about 5 pM to about 450 pM, about 5 pM to about 400 pM, about 5 pM to about 3 ...500 pM, about 5 pM to about 500 pM, about 5 pM to about 500 pM, about 5 M to about 300 pM, about 5 pM to about 250 pM, about 5 pM to about 200 pM, about 5 pM to about 150 pM, about 5 pM to about 100 pM, about 5 pM to about 90 pM, about 5 pM to about 80 pM, about 5 pM to about 70 pM, about 5 pM to about 60 pM, about 5 pM to about 50 pM, about 5 pM to about 40 pM, about 5pM to about 30pM, about 5pM to about 20pM, about 5pM to about 10pM, about 10pM to about 30nM, about 10pM to about 25nM, about 10pM to about 20nM, about 10pM to about 15nM, about 10pM to about 10nM, about 10pM to about 5nM, about 10pM to about 2nM, about 10pM to about 1nM , about 10 pM to about 950 pM, about 10 pM to about 900 pM, about 10 pM to about 850 pM, about 10 pM to about 800 pM, about 10 pM to about 750 pM, about 10 pM to about 700 pM, about 10 pM to about 650 pM, about 10 pM to about 600 pM, about 10 pM to about 550 pM, about 10 pM to about about 500 pM, about 10 pM to about 450 pM, about 10 pM to about 400 pM, about 10 pM to about 350 pM, about 10 pM to about 300 pM, about 10 pM to about 250 pM, about 10 pM to about 200 pM, about 10 pM to about 150 pM, about 10 pM to about 100 pM, about 10 pM to about 90 pM, about 10 pM to about 80 pM, about 10 pM to about 70 pM, about 10 pM to about 60 pM, about 10 pM to about 50 pM, about 10 pM to about 40 pM, about 10 pM to about 30 pM, about 10 pM to about 20 pM, about 15 pM to about 30 nM, about 15 pM to about 25 nM, about 15 pM to about 20 nM, about 15 pM to about 25 nM M to about 15 nM, about 15 pM to about 10 nM, about 15 pM to about 5 nM, about 15 pM to about 2 nM, about 15 pM to about 1 nM, about 15 pM to about 950 pM, about 15 pM to about 900 pM, about 15 pM to about 850 pM, about 15 pM to about 800 pM, about 15 pM to about 750 pM, about 15 pM to about about 700 pM, about 15 pM to about 650 pM, about 15 pM to about 600 pM, about 15 pM to about 550 pM, about 15 pM to about 500 pM, about 15 pM to about 450 pM, about 15 pM to about 400 pM, about 15 pM to about 350 pM, about 15 pM to about 300 pM, about 15 pM to about 250 pM,about 15 pM to about 200 pM, about 15 pM to about 150 pM, about 15 pM to about 100 pM, about 15 pM to about 90 pM, about 15 pM to about 80 pM, about 15 pM to about 70 pM, about 15 pM to about 60 pM, about 15 pM to about 50 pM, about 15 pM to about 40 pM, about 15 pM to about 30 pM, about 15pM to about 20pM, about 20pM to about 30nM, about 20pM to about 25nM, about 20pM to about 20nM, about 20pM to about 15nM, about 20pM to about 10nM, about 20pM to about 5nM, about 20pM to about 2nM, about 20pM to about 1nM, about 20pM to about 950pM, about 20pM to about 900 pM, about 20 pM to about 850 pM, about 20 pM to about 800 pM, about 20 pM to about 750 pM, about 20 pM to about 700 pM, about 20 pM to about 650 pM, about 20 pM to about 600 pM, about 20 pM to about 550 pM, about 20 pM to about 500 pM, about 20 pM to about 450 pM, about 20 pM to about 400 pM, about 20 pM to about 350 pM, about 20 pM to about 300 pM, about 20 pM to about 250 pM, about 20 pM to about 20 pM, about 200 pM to about 150 pM, about 20 pM to about 100 pM, about 20 pM to about 90 pM, about 20 pM to about 80 pM, about 20 pM to about 70 pM M, about 20 pM to about 60 pM, about 20 pM to about 50 pM, about 20 pM to about 40 pM, about 20 pM to about 30 pM, about 30 pM to about 30 nM, about 30 pM to about 25 nM, about 30 pM to about 30 nM, about 30 pM to about 15 nM, about 30 pM to about 10 nM, about 30 pM to about 5 nM, about 30 pM to about 2 nM, about 30 pM to about 1 nM, about 30 pM to about 950 pM, about 30 pM to about 900 pM, about 30 pM to about 850 pM, about 30 pM to about 800 pM, about 30 pM to about 750 pM, about 30 pM to about 700 pM, about 30 pM to about 650 pM, about 30 pM to about 600 pM, about 30 pM to about 550 pM, about 30 pM to about 500 pM, about 30 pM to about 450 pM, about 30 pM to about 400 pM, about 30 pM to about 350 pM, about 30 pM to about 300 pM, about 30 pM to about 250 pM, about 30 pM to about 200 pM, about 30 pM to about 150 pM, about 30 pM to about 1 00 pM, about 30 pM to about 90 pM, about 30 pM to about 80 pM, about 30 pM to about 70 pM, about 30 pM to about 60 pM, about 30 pM to about 50 pM, about 30 pM to about 40 pM, about 40 pM to about 30 nM, about 40 pM to about 25 nM, about 40 pM to about 30 nM, about 40 pM to about 15 nM,about 40 pM to about 10 nM, about 40 pM to about 5 nM, about 40 pM to about 2 nM, about 40 pM to about 1 nM, about 40 pM to about 950 pM, about 40 pM to about 900 pM, about 40 pM to about 850 pM, about 40 pM to about 800 pM, about 40 pM to about 750 pM, about 40 pM to about 700 pM, about 40 pM to about 650 pM, about 40 pM to about 600 pM, about 40 pM to about 550 pM, about 40 pM to about 500 pM, about 40 pM to about 450 pM, about 40 pM to about 400 pM, about 40 pM to about 350 pM, about 40 pM to about 300 pM, about 40 pM to about 250 pM, about 40 pM to about 200 pM, about 40 pM to about 150 pM, about 40 pM to about 100 pM, about 40 pM to about 90 pM, about 40 pM to about 80 pM, about 40 pM to about 70 pM, about 40 pM to about 60 pM, about 40 pM to about 50 pM, about 50 pM to about 30 nM, about 50 pM to about 25 nM, about 50 pM to about 30nM, about 50pM to about 15nM, about 50pM to about 10nM, about 50pM to about 5nM, about 50pM to about 2nM, about 50pM to about 1nM, about 50pM to about 950pM, about 50pM to about 900pM, about 50pM to about 850pM, about 50pM to about 800pM, about 50pM to about 750 pM, about 50 pM to about 700 pM, about 50 pM to about 650 pM, about 50 pM to about 600 pM, about 50 pM to about 550 pM, about 50 pM to about 500 pM, about 50 pM to about 450 pM, about 50 pM to about 400 pM, about 50 pM to about 350 pM, about 50 pM to about 300 pM, about 50 pM to about 250 pM, about 50 pM to about 200 pM, about 50 pM to about 150 pM, about 50 pM to about 100 pM, about 50 pM to about 90 pM, about 50 pM to about 80 pM, about 50 pM to about 70 pM, about 50 pM to about 60 pM, about 60 pM to about 30 nM, about 60 pM to about 25 nM, about 60 pM to about 30 nM, about 60 pM to about 15 nM, about 60 pM to about 10 nM, about 60 pM to about 5 nM, about 60 pM to about 2 nM, about 60 pM to about 1 nM, about 60 pM to about 950 pM, about 60 pM to about 900 pM, about 60 pM to about 850 pM, about 60 pM to about 800 pM, about 60 pM to about about 750 pM, about 60 pM to about 700 pM, about 60 pM to about 650 pM, about 60 pM to about 600 pM, about 60 pM to about 550 pM, about 60 pM to about 500 pM, about 60 pM to about 450 pM, about 60 pM to about 400 pM, about 60 pM to about 350 pM, about 60 pM to about 300 pM,about 60 pM to about 250 pM, about 60 pM to about 200 pM, about 60 pM to about 150 pM, about 60 pM to about 100 pM, about 60 pM to about 90 pM, about 60 pM to about 80 pM, about 60 pM to about 70 pM, about 70 pM to about 30 nM, about 70 pM to about 25 nM, about 70 pM to about 30 nM, about 70 pM to about 15 nM, about 70 pM to about 10 nM, about 70 pM to about 5 nM, about 70 pM to about 2 nM, about 70 pM to about 1 nM, about 70 pM to about 950 pM, about 70 pM to about 900 pM, about 70 pM to about 850 pM, about 70 pM to about 800 pM, about 70 pM to about 750 pM, about 70 pM pM to about 700 pM, about 70 pM to about 650 pM, about 70 pM to about 600 pM, about 70 pM to about 550 pM, about 70 pM to about 500 pM, about 70 pM to about 450 pM, about 70 pM to about 400 pM, about 70 pM to about 350 pM, about 70 pM to about 300 pM, about 70 pM to about 250 pM , about 70 pM to about 200 pM, about 70 pM to about 150 pM, about 70 pM to about 100 pM, about 70 pM to about 90 pM, about 70 pM to about 80 pM, about 80 pM to about 30 nM, about 80 pM to about 25 nM, about 80 pM to about 30 nM, about 80 pM to about 15 nM, about 80 pM to about 10 nM, about 80 pM to about 5 nM, about 80 pM to about 2 nM, about 80 pM to about 1 nM, about 80 pM to about 950 pM, about 80 pM to about 900 pM, about 80 pM to about 850 pM, about 80 pM to about 800 pM, about 80 pM to about 750 pM, about 80 pM to about 700 pM, about 80 pM to about 650 pM, about 8 0 pM to about 600 pM, about 80 pM to about 550 pM, about 80 pM to about 500 pM, about 80 pM to about 450 pM, about 80 pM to about 400 pM, about 80 pM to about 350 pM, about 80 pM to about 300 pM, about 80 pM to about 250 pM, about 80 pM to about 200 pM, about 80 pM to about 150 pM, about 80 pM to about 100 pM, about 80 pM to about 90 pM, about 90 pM to about 30 nM, about 90 pM to about 25 nM, about 90 pM to about 30 nM, about 90 pM to about 15 nM, about 90 pM to about 10 nM, about 90 pM to about 5 nM, about 90 pM to about 2 nM, about 90 pM to about 1 nM, about 90 pM to about pM to about 950 pM, about 90 pM to about 900 pM, about 90 pM to about 850 pM, about 90 pM to about 800 pM, about 90 pM to about 750 pM, about 90 pM to about 700 pM, about 90 pM to about 650 pM, about 90 pM to about 600 pM, about 90 pM to about 550 pM, about 90 pM to about 500 pM,about 90pM to about 450pM, about 90pM to about 400pM, about 90pM to about 350pM, about 90pM to about 3, 00 pM, about 90 pM to about 250 pM, about 90 pM to about 200 pM, about 90 pM to about 150 pM, about 90 pM to about 100 pM, about 100 pM to about 30 nM, about 100 pM to about 25 nM, about 100 pM to about 30 nM, about 100 pM to about 15 nM, about 100 pM to about 10 nM, about 100 pM to about 5 nM, about 100 pM to about 2 nM, about 100 pM to about 1 nM, about 100 pM to about 950 pM, about 100 pM to about 900 pM, about 100 pM to about 850 pM, about 100 pM to about 800 pM, about 100 pM to about 750 pM, about 100 pM to about 700 pM, about 100 pM to about M to about 650 pM, about 100 pM to about 600 pM, about 100 pM to about 550 pM, about 100 pM to about 500 pM, about 100 pM to about 450 pM, about 100 pM to about 400 pM, about 100 pM to about 350 pM, about 100 pM to about 300 pM, about 100 pM to about 250 pM, about 10 0 pM to about 200 pM, about 100 pM to about 150 pM, about 150 pM to about 30 nM, about 150 pM to about 25 nM, about 150 pM to about 30 nM, about 150 pM to about 15 nM, about 150 pM to about 10 nM, about 150 pM to about 5 nM, about 150 pM to about 2 nM, about 150 pM to about 1 nM M, about 150 pM to about 950 pM, about 150 pM to about 900 pM, about 150 pM to about 850 pM, about 150 pM to about 800 pM, about 150 pM to about 750 pM, about 150 pM to about 700 pM, about 150 pM to about 650 pM, about 150 pM to about 600 pM, about 150 pM to about 5 50pM, about 150pM to about 500pM, about 150pM to about 450pM, about 150pM to about 400pM, about 150pM to about 350pM, about 150pM to about 300pM, about 150pM to about 250pM, about 150pM to about 200pM, about 200pM to about 30nM, about 200pM to about 25nM, about 200pM to about 30nM, about 200pM to about 15nM, about 200pM to about 10nM, about 200pM to about 5nM, about 200pM to about 2nM, about 200pM to about 1nM, about 200pM to about 950pM, about 200pM to about 900pM, about 200pM to about 850pM, about 2 00 pM to about 800 pM, about 200 pM to about 750 pM, about 200 pM to about 700 pM, about 200 pM to about 650 pM, about 200 pM to about 600 pM, about 200 pM to about 550 pM, about 200 pM to about 500 pM, about 200 pM to about 450 pM, about 200 pM to about 400 pM,about 200 pM to about 350 pM, about 200 pM to about 300 pM, about 200 pM to about 250 pM, about 300 pM to about 30 nM, about 300 pM to about 25 nM, about 300 pM to about 30 nM, about 300 pM to about 15 nM, about 300 pM to about 10 nM, about 300 pM to about 5 nM, about 300 pM to about 5 nM, about 300 pM to about 15 nM, about 300 pM to about 10 nM, about 300 pM to about 5 nM, about 300 pM to about 1 ...0 nM, about 300 pM to about 5 nM, about 2 nM to about 1 nM, about 300 pM to about 950 pM, about 300 pM to about 900 pM, about 300 pM to about 850 pM, about 300 pM to about 800 pM, about 300 pM to about 750 pM, about 300 pM to about 700 pM, about 300 pM to about 650 pM, about 300 pM to about 1000 pM, about 300 pM to about 1500 pM, about 300 pM to about 2000 pM, about 300 pM to about 35 ... about 600 pM, about 300 pM to about 550 pM, about 300 pM to about 500 pM, about 300 pM to about 450 pM, about 300 pM to about 400 pM, about 300 pM to about 350 pM, about 400 pM to about 30 nM, about 400 pM to about 25 nM, about 400 pM to about 30 nM, about 400 pM to about 15nM, about 400pM to about 10nM, about 400pM to about 5nM, about 400pM to about 2nM, about 400pM to about 1nM, about 400pM to about 950pM, about 400pM to about 900pM, about 400pM to about 850pM, about 400pM to about 800pM, about 400pM to about 750pM, about 400 pM to about 700 pM, about 400 pM to about 650 pM, about 400 pM to about 600 pM, about 400 pM to about 550 pM, about 400 pM to about 500 pM, about 500 pM to about 30 nM, about 500 pM to about 25 nM, about 500 pM to about 30 nM, about 500 pM to about 15 nM, about 5 00 pM to about 10 nM, about 500 pM to about 5 nM, about 500 pM to about 2 nM, about 500 pM to about 1 nM, about 500 pM to about 950 pM, about 500 pM to about 900 pM, about 500 pM to about 850 pM, about 500 pM to about 800 pM, about 500 pM to about 750 pM, about 500 pM to about 1000 pM, about 500 pM to about 1000 pM, about 500 pM to about 2000 pM, about 500 pM to about 1 ... about 700 pM, about 500 pM to about 650 pM, about 500 pM to about 600 pM, about 500 pM to about 550 pM, about 600 pM to about 30 nM, about 600 pM to about 25 nM, about 600 pM to about 30 nM, about 600 pM to about 15 nM, about 600 pM to about 10 nM, about 600 pM to about 5 nM M, about 600 pM to about 2 nM, about 600 pM to about 1 nM, about 600 pM to about 950 pM, about 600 pM to about 900 pM, about 600 pM to about 850 pM, about 600 pM to about 800 pM, about 600 pM to about 750 pM, about 600 pM to about 700 pM, about 600 pM to about 650 pM,Approximately 700pM to approximately 30nM, approximately 700pM to approximately 25nM, approximately 700pM to approximately 30nM, approximately 700pM to approximately 15nM, approximately 700pM to approximately 10nM, approximately 700pM to approximately 5nM, approximately 700pM to approximately 2nM, approximately 700pM to approximately 1 nM, about 700 pM to about 950 pM, about 700 pM to about 900 pM, about 700 pM to about 850 pM, about 700 pM to about 800 pM, about 700 pM to about 750 pM, about 800 pM to about 30 nM, about 800 pM to about 25 nM, Approximately 800pM to approximately 30nM, approximately 800pM to approximately 15nM, approximately 800pM to approximately 10nM, approximately 800pM to approximately 5nM, approximately 800pM to approximately 2nM, approximately 800pM to approximately 1nM, approximately 800pM to approximately 950pM, approximately 800pM to approximately 90 0pM, about 800pM to about 850pM, about 900pM to about 30nM, about 900pM to about 25nM, about 900pM to about 30nM, about 900pM to about 15nM, about 900pM to about 10nM, about 900pM to about 5nM, about 900 pM to about 2nM, about 900pM to about 1nM, about 900pM to about 950pM, about 1nM to about 30nM, about 1nM to about 25nM, about 1nM to about 20nM, about 1nM to about 15nM, about 1nM to about 10nM, about 1nM to about 5nM , about 2nM to about 30nM, about 2nM to about 25nM, about 2nM to about 20nM, about 2nM to about 15nM, about 2nM to about 10nM, about 2nM to about 5nM, about 4nM to about 30nM, about 4nM to about 25nM, about 4nM to about 20nM, about 4 nM to about 15 nM, about 4 nM to about 10 nM, about 4 nM to about 5 nM, about 5 nM to about 30 nM, about 5 nM to about 25 nM, about 5 nM to about 20 nM, about 5 nM to about 15 nM, about 5 nM to about 10 nM, about 10 nM to about 30 nM, about 10 nM to about 25 nM, about 10 nM to about 20 nM, about 10 nM to about 15 nM, about 15 nM to about 30 nM, about 15 nM to about 25 nM, about 15 nM to about 20 nM, about 20 nM to about 30 nM, and about 20 nM to about 25 nM).

[0081] Any of the target binding domains described herein may be administered at a concentration of about 1 nM to about 10 nM (e.g., about 1 nM to about 9 nM, about 1 nM to about 8 nM, about 1 nM to about 7 nM, about 1 nM to about 6 nM, about 1 nM to about 5 nM, about 1 nM to about 4 nM, about 1 nM to about 3 nM, about 1 nM to about 2 nM, about 2 nM to about 10 nM, about 2 nM to about 9 nM, about 2 nM to about 8 nM, about 2 nM to about 7 nM, about 2 nM to about 6 nM, about 2 nM to about 5 nM, about 2 nM to about 4 nM, about 2 nM to about 3 nM, about 3 nM to about 10 nM, about 3 nM to about 9 nM, about 3 nM to about 8 nM, about 3 nM to about 7 nM, about 3 nM to about Approximately 6nM, approximately 3nM to approximately 5nM, approximately 3nM to approximately 4nM, approximately 4nM to approximately 10nM, approximately 4nM to approximately 9nM, approximately 4nM to approximately 8nM, approximately 4nM~ Approximately 7nM, approximately 4nM to approximately 6nM, approximately 4nM to approximately 5nM, approximately 5nM to approximately 10nM, approximately 5nM to approximately 9nM, approximately 5nM to approximately 8nM, approximately 5nM to Approximately 7nM, approximately 5nM to approximately 6nM, approximately 6nM to approximately 10nM, approximately 6nM to approximately 9nM, approximately 6nM to approximately 8nM, approximately 6nM to approximately 7nM, approximately 7nM~ about 10 nM, about 7 nM to about 9 nM, about 7 nM to about 8 nM, about 8 nM to about 10 nM, about 8 nM to about 9 nM, and about 9 nM to about 10 nM) D It can bind to the ligand of TGFβRII having the formula:

[0082] A variety of different methods known in the art can be used to quantify the K of any of the antigen binding protein constructs described herein. D Values ​​can be determined (eg, electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance, biomolecular binding kinetics assays, etc.).

[0083] antigen-binding domain In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

[0084] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to different antigens.

[0085] In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain are antigen-binding domains. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each antigen-binding domains.

[0086] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the antigen-binding domain comprises or is an scFv or a single domain antibody (e.g., a VHH or VNAR domain).

[0087] In some examples, the antigen binding domain (e.g., any of the antigen binding domains described herein) can specifically bind to a ligand of TGF-βRII (see, e.g., the antigen binding domains capable of specifically binding to TGF-β described in US 2021 / 0061897, US 2020 / 0399358, US 2020 / 0392221, US 2019 / 0315850, and US 2019 / 0177406, each of which is incorporated herein by reference).

[0088] Each antigen-binding domain present in any of the multi-chain chimeric polypeptides described herein is independently selected from the group consisting of a VHH domain, a VNAR domain, and an scFv. In some embodiments, any of the antigen-binding domains described herein is a BiTe, (scFv)2, a nanobody, a nanobody-HSA, a DART, a TandAb, a scdiabody, a scdiabody-CH3, a scFv-CH-CL-scFv, an HSAbody, a scdiabody-HAS, or a tandem-scFv. Further examples of antigen-binding domains that can be used in any of the multi-chain chimeric polypeptides are known in the art.

[0089] VHH domains are single monomeric variable antibody domains that can be found in camelids. VNAR domains are single monomeric variable antibody domains that can be found in cartilaginous fish. VHH domains and V NARNon-limiting aspects of the domain are described, for example, in Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016; De Genst et al., Dev. Comp. Immunol. 30:187-198, 2006; De Meyer et al., Trends Biotechnol. 32:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174, 2015; Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014; Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016; Mujic-Delic et al., Trends Pharmacol. Sci. 35:247-255, 2014; Muyldermans, J. Biotechnol. 74:277-302, 2001; Muyldermans et al., Trends Biochem. Sci. 26:230-235, 2001; Muyldermans, Ann. Rev. Biochem. 8:775-797, 2013; Rahbarizadeh et al., Immunol. Invest. 40:299-338, 2011; Van Audenhove et al., EBioMedicine 8:40-48, 2016; Van Bockstaele et al., Curr. Opin. Investig. Drugs 10:1212-1224, 2009; Vincke et al., Methods Mol. Biol. 911:15-26, 2012, and Wesolowski et al., Med. Microbiol. Immunol. 198:157-174, 2009.

[0090] In some embodiments, each of the antigen-binding domains in the multi-chain chimeric polypeptides described herein are both VHH domains, or at least one antigen-binding domain is a VHH domain. In some embodiments, each of the antigen-binding domains in the multi-chain chimeric polypeptides described herein are both VNAR domains, or at least one antigen-binding domain is a VNAR domain. In some embodiments, each of the antigen-binding domains in the multi-chain chimeric polypeptides described herein are both scFv domains, or at least one antigen-binding domain is an scFv domain.

[0091] In some embodiments, two or more of the polypeptides present in the multi-chain chimeric polypeptide are any of the antigen-binding domains described herein, e.g., an antigen-binding fragment of an antibody (e.g., any of the antigen-binding fragments of an antibody described herein), VHH-scAb, VHH-Fab, Dual scFab, F(ab')2, diabody, crossMab, DAF (two in one), DAF (four in one), DutaMab, DT-IgG, knobs-in-hole common light chain, knobs-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2 They can be assembled (e.g., non-covalently assembled) to form scFv, scFv 4-Ig, Zybody, DVI-IgG, diabody-CH3, triplebody, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody, dock and lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, and scFv 1-PEG-scFv2. See, e.g., Spiess et al., Mol. Immunol. 67:95-106, 2015, which is incorporated herein in its entirety for a description of these elements. Non-limiting examples of antigen-binding fragments of antibodies include Fv fragments, Fab fragments, F(ab')2 fragments, and Fab' fragments.Further examples of antigen-binding fragments of antibodies are antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, IgG2, IgG3, or IgG4) (e.g., antigen-binding fragments of human or humanized IgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4); antigen-binding fragments of IgA (e.g., antigen-binding fragments of IgA1 or IgA2) (e.g., antigen-binding fragments of human or humanized IgA, e.g., human or humanized IgA1 or IgA2); antigen-binding fragments of IgD (e.g., antigen-binding fragments of human or humanized IgD); antigen-binding fragments of IgE (e.g., antigen-binding fragments of human IgE or humanized IgE); or antigen-binding fragments of IgM (e.g., antigen-binding fragments of human or humanized IgM).

[0092] An "Fv" fragment comprises a non-covalent dimer of one heavy- and one light-chain variable domain.

[0093] A "Fab" fragment contains the heavy and light chain variable domains of an Fv fragment as well as the constant domain of the light chain and the first constant domain of the heavy chain (C H1 ) is included.

[0094] An "F(ab')2" fragment contains two Fab fragments linked by a disulfide bond near the hinge region.

[0095] "Dual variable domain immunoglobulin" or "DVD-Ig" refers to multivalent and multispecific binding proteins described, for example, in DiGiammarino et al., Methods Mol. Biol. 899:145-156, 2012; Jakob et al., MABs 5:358-363, 2013; and U.S. Patent Nos. 7,612,181; 8,258,268; 8,586,714; 8,716,450; 8,722,855; 8,735,546; and 8,822,645, each of which is incorporated by reference in its entirety.

[0096] DART is described, for example, in Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

[0097] In some embodiments, any of the antigen binding domains described herein can bind to an antigen selected from the group consisting of proteins, carbohydrates, lipids, and combinations thereof.

[0098] Further examples and embodiments of antigen-binding domains are known in the art.

[0099] Soluble receptors In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin receptor, a soluble cytokine receptor, or a ligand receptor. In some embodiments, the soluble receptor is a soluble TGF-β receptor II (TGF-βRII) (see, e.g., as described in Yung et al., Am. J. Resp. Crit. Care Med. 194(9):1140-1151, 2016) or a soluble TGF-βRIII (see, e.g., as described in Heng et al., Placenta 57:320, 2017).

[0100] Further examples of soluble interleukin receptors and soluble cytokine receptors are known in the art.

[0101] Additional target binding domains In some embodiments of any of the multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art), wherein at least one of the one or more additional antigen binding domains is positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) and the first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein). In some embodiments, the first chimeric polypeptide may further comprise a linker sequence (e.g., any of the exemplary linker sequences described herein) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein), and / or a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and a first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein of the exemplary affinity domain pairs described herein).

[0102] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains at the N-terminus and / or C-terminus of the first chimeric polypeptide. In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is directly adjacent to the first domain of a pair of affinity domains in the first chimeric polypeptide (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art).

[0103] In some embodiments of any of the multi-chain chimeric polypeptides described herein, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is disposed at the N- and / or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) and the first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) of the one or more additional target binding domains disposed at the N-terminus is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the linker sequences described herein or known in the art) disposed between the at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) in the first chimeric polypeptide and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein).In some embodiments, at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) of the one or more additional target binding domains disposed at the C-terminus is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the linker sequences described herein or known in the art) disposed between the at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) in the first chimeric polypeptide and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) located between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the pair of affinity domains (e.g., any of the first domains described herein or any of the exemplary affinity domain pairs described herein) is immediately adjacent to the soluble tissue factor domain and / or the first domain of the pair of affinity domains.In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary soluble tissue factor domains described herein) disposed between (i) the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and at least one of one or more additional target binding domains (e.g., exemplary target binding domains described herein or known in the art), located between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary affinity domain pairs described herein); and / or (ii) between the first domain of the pair of affinity domains and at least one of the one or more additional target binding domains, located between the first domain of the pair of affinity domains and the soluble tissue factor domain and the first domain of the pair of affinity domains.

[0104] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any of the exemplary target binding domains described herein) at the N-terminus and / or C-terminus of the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is immediately adjacent to the second domain of a pair of affinity domains in the second chimeric polypeptide (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein). In some embodiments, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) in the second chimeric polypeptide and the second domain of the pair of affinity domains (e.g., any of the second domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is directly adjacent to the second target binding domain (e.g., any of the target binding domains described herein or known in the art) in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) in the second chimeric polypeptide and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art).

[0105] In some embodiments of any of the multi-chain chimeric polypeptides described herein, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same antigen. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same epitope. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each comprise the same amino acid sequence.

[0106] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to different antigens. In some aspects of any of the multi-chain chimeric polypeptides described herein, one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target binding domain, the second target binding domain, and the one or more additional target binding domains are antigen binding domains. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains are each antigen binding domains (e.g., scFvs or single-domain antibodies).

[0107] Affinity domain pair In some embodiments, the multi-chain chimeric polypeptide comprises 1) a first chimeric polypeptide comprising a first domain of a pair of affinity domains, and 2) a second chimeric polypeptide comprising a second domain of a pair of affinity domains, such that the first chimeric polypeptide and the second chimeric polypeptide associate through binding of the first and second domains of the pair of affinity domains. In some embodiments, the pair of affinity domains is a sushi domain derived from the alpha chain of the human IL-15 receptor (IL15Rα) and soluble IL-15. The sushi domain, also known as the short consensus repeat or type 1 glycoprotein motif, is a common motif in protein-protein interactions. Sushi domains have been identified in many protein-binding molecules, including complement components C1r, C1s, factor H, and C2m, as well as the non-immunological molecules factor XIII and β2-glycoprotein. A typical sushi domain has approximately 60 amino acid residues and contains four cysteines (Ranganathan, Pac. Symp. Biocomput. 2000:155-67). The first cysteine ​​can form a disulfide bond with the third cysteine, and the second cysteine ​​can form a disulfide bridge with the fourth cysteine. In some embodiments where one member of the affinity domain pair is soluble IL-15, the soluble IL15 has a D8N or D8A amino acid substitution. In some embodiments where one member of the affinity domain pair is the alpha chain of the human IL-15 receptor (IL15Rα), the human IL15Rα is mature full-length IL15Rα. In some embodiments, the affinity domain pair is barnase and barnstar. In some embodiments, the affinity domain pair is PKA and AKAP.In some embodiments, the affinity domain pair is an adapter / docking tag module based on a mutant RNase I fragment (Rossi, Proc Natl Acad Sci USA. 103:6841-6846, 2006; Sharkey et al., Cancer Res. 68:5282-5290, 2008; Rossi et al., Trends Pharmacol Sci. 33:474-481, 2012) or a SNARE module based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25 (Deyev et al., Nat Biotechnol. 1486-1492, 2003).

[0108] In some embodiments, a first chimeric polypeptide of the multi-chain chimeric polypeptide comprises a first domain of a pair of affinity domains and a second chimeric polypeptide of the multi-chain chimeric polypeptide comprises a second domain of the pair of affinity domains, wherein the first domain of the pair of affinity domains and the second domain of the pair of affinity domains have a dissociation equilibrium constant (K D ) is 1×10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, 1 x 10 -10 Less than M, 1 x 10 -11 Less than M, 1 x 10 -12 Less than M or 1 x 10 -13 In some embodiments, the first domain of the pair of affinity domains and the second domain of the pair of affinity domains bind to each other with less than K D But about 1x10 -4 M ~ approx. 1x10 -6 M, about 1x10 -5 M ~ approx. 1x10 -7 M, about 1x10 -6 M ~ approx. 1x10 -8 M, about 1x10 -7 M ~ approx. 1x10 -9 M, about 1x10 -8 M ~ approx. 1x10 -10 M, about 1x10 -9 M ~ approx. 1x10 -11 M, about 1x10-10 M ~ approx. 1x10 -12 M, about 1x10 -11 M ~ approx. 1x10 -13 M, about 1x10 -4 M ~ approx. 1x10 -5 M, about 1x10 -5 M ~ approx. 1x10 -6 M, about 1x10 -6 M ~ approx. 1x10 -7 M, about 1x10 -7 M ~ approx. 1x10 -8 M, about 1x10 -8 M ~ approx. 1x10 -9 M, about 1x10 -9 M ~ approx. 1x10 -10 M, about 1x10 -10 M ~ approx. 1x10 -11 M, about 1x10 -11 M ~ approx. 1x10 -12 M, or approximately 1x10 -12 M ~ approx. 1x10 -13 The K of binding between the first domain of the pair of affinity domains and the second domain of the pair of affinity domains can be determined using any of a variety of different methods known in the art. D Values ​​can be determined (eg, electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance and biomolecular binding kinetic assays, etc.).

[0109] In some embodiments, a first chimeric polypeptide of the multi-chain chimeric polypeptide comprises a first domain of a pair of affinity domains, and a second chimeric polypeptide of the multi-chain chimeric polypeptide comprises a second domain of the pair of affinity domains, wherein the first domain of the pair of affinity domains, the second domain of the pair of affinity domains, or both, are about 10 to 100 amino acids in length. For example, the first domain of the pair of affinity domains, the second domain of the pair of affinity domains, or both may be about 10 to 100 amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to 100 amino acids in length, about 10 to 95 amino acids in length, about 10 to 90 amino acids in length, about 10 to 85 ... The length may be 0 amino acids, about 10 to 75 amino acids, about 10 to 70 amino acids, about 10 to 65 amino acids, about 10 to 60 amino acids, about 10 to 55 amino acids, about 10 to 50 amino acids, about 10 to 45 amino acids, about 10 to 40 amino acids, about 10 to 35 amino acids, about 10 to 30 amino acids, about 10 to 25 amino acids, about 10 to 20 amino acids, about 10 to 15 amino acids, about 20 to 30 amino acids, about 30 to 40 amino acids, about 40 to 50 amino acids, about 50 to 60 amino acids, about 60 to 70 amino acids, about 70 to 80 amino acids, about 80 to 90 amino acids, about 90 to 100 amino acids, about 20 to 90 amino acids, about 30 to 80 amino acids, about 40 to 70 amino acids, about 50 to 60 amino acids, or any range therebetween. In some embodiments, the first domain of the pair of affinity domains, the second domain of the pair of affinity domains, or both, are about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

[0110] In some embodiments, any of the first and / or second domains of a pair of affinity domains disclosed herein can include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at its N-terminus and / or C-terminus, so long as the function of the first and / or second domain of the pair of affinity domains remains intact. For example, the sushi domain derived from the alpha chain of the human IL-15 receptor (IL15Rα) can include one or more additional amino acids at its N-terminus and / or C-terminus while still retaining the ability to bind to soluble IL-15. Additionally, or alternatively, soluble IL-15 can include one or more additional amino acids at its N-terminus and / or C-terminus while still retaining the ability to bind to the sushi domain derived from the alpha chain of the human IL-15 receptor (IL15Rα).

[0111] Non-limiting examples of sushi domains from the alpha chain of IL-15 receptor alpha (IL15Rα) can include a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 16). In some embodiments, the sushi domain from the alpha chain of IL15Rα can be encoded by a nucleotide sequence comprising ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 17).

[0112] In some embodiments, the soluble IL-15 may comprise a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 18). In some embodiments, soluble IL-15 can be encoded by a nucleic acid comprising the sequence AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 19).

[0113] In some embodiments, soluble IL-15 can comprise a D8N amino acid substitution. In some embodiments, soluble IL-15 with the D8N variant (IL15D8N) can comprise a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to NWVNVISNLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 70). In some embodiments, soluble IL-15 having the D8N mutant (IL15D8N) can be encoded by a nucleic acid comprising the sequence AACTGGGTGAATGTAATAAGTAATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT (SEQ ID NO: 71).

[0114] signal sequence In some embodiments, a multi-chain chimeric polypeptide comprises a first chimeric polypeptide comprising a signal sequence at its N-terminus. In some embodiments, a multi-chain chimeric polypeptide comprises a second chimeric polypeptide comprising a signal sequence at its N-terminus. In some embodiments, both the first chimeric polypeptide of a multi-chain chimeric polypeptide and the second chimeric polypeptide of a multi-chain chimeric polypeptide comprise a signal sequence. As will be understood by those skilled in the art, a signal sequence is an amino acid sequence present at the N-terminus of many endogenously produced proteins that directs the protein into the secretory pathway (e.g., directing the protein to reside in a specific intracellular organelle, to reside at the plasma membrane, or to be secreted from the cell). Signal sequences are heterogeneous and vary greatly in their primary amino acid sequence. However, signal sequences are typically 16-30 amino acids in length and comprise a hydrophilic, usually positively charged, N-terminal region, a central hydrophobic domain, and a C-terminal region containing a cleavage site for a signal peptidase.

[0115] In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprises a signal sequence having the amino acid sequence MKWVTFISLLFLFSSAYS (SEQ ID NO: 20). In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprises a signal sequence encoded by the sequence ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO: 21), ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC (SEQ ID NO: 22), or ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO: 23).

[0116] In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprise a single sequence having the amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 24). In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprise a single sequence having the amino acid sequence MGQIVTMFEALPHIIDEVINIVIIVLIIITSIKAVYNFATCGILALVSFLFLAGRSCG (SEQ ID NO: 25). In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprise a single sequence having the amino acid sequence MPNHQSGSPTGSSDLLLSGKKQRPHLALRRKRRREMRKINRKVRRMNLAPIKEKTAWQHLQALISEAEEVLKTSQTPQNSLTLFLALLSVLGPPVTG (SEQ ID NO: 26). In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprise a single sequence having the amino acid sequence MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO: 27). One of skill in the art will recognize other suitable signal sequences for use in the first chimeric polypeptide and / or second chimeric polypeptide of the multi-chain chimeric polypeptides described herein.

[0117] In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprises a signal sequence that is about 10 to 100 amino acids in length. For example, the single sequence may be about 10 to 100 amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to 100 amino acids in length, about 10 to 95 amino acids in length, about 10 to 90 amino acids in length, about 10 to 85 amino acids in length, about 10 to 80 amino acids in length, about 10 to 75 amino acids in length, The length may be about 10 to 70 amino acids, about 10 to 65 amino acids, about 10 to 60 amino acids, about 10 to 55 amino acids, about 10 to 50 amino acids, about 10 to 45 amino acids, about 10 to 40 amino acids, about 10 to 35 amino acids, about 10 to 30 amino acids, about 10 to 25 amino acids, about 10 to 20 amino acids, about 10 to 15 amino acids, about 20 to 30 amino acids, about 30 to 40 amino acids, about 40 to 50 amino acids, about 50 to 60 amino acids, about 60 to 70 amino acids, about 70 to 80 amino acids, about 80 to 90 amino acids, about 90 to 100 amino acids, about 20 to 90 amino acids, about 30 to 80 amino acids, about 40 to 70 amino acids, about 50 to 60 amino acids, or any range therebetween. In some embodiments, the signal sequence is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

[0118] In some embodiments, any of the signal sequences disclosed herein can include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at its N-terminus and / or C-terminus, as long as the function of the signal sequence remains intact. For example, a signal sequence having the amino acids MKCLLYLAFLFLGVNC (SEQ ID NO: 28) can include one or more additional amino acids at the N-terminus or C-terminus while still retaining the function of directing a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of a multi-chain chimeric polypeptide, or both, into the secretory pathway.

[0119] In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprises a signal sequence that directs the multi-chain chimeric polypeptide to the extracellular space. Such embodiments are useful for producing multi-chain chimeric polypeptides that are relatively easy to isolate and / or purify.

[0120] Peptide tags In some embodiments, a multi-chain chimeric polypeptide comprises a first chimeric polypeptide comprising a peptide tag (e.g., at the N-terminus or C-terminus of the first chimeric polypeptide). In some embodiments, a multi-chain chimeric polypeptide comprises a second chimeric polypeptide comprising a peptide tag (e.g., at the N-terminus or C-terminus of the second chimeric polypeptide). In some embodiments, both the first chimeric polypeptide of the multi-chain chimeric polypeptide and the second chimeric polypeptide of the multi-chain chimeric polypeptide comprise a peptide tag. In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both comprise two or more peptide tags.

[0121] Exemplary peptide tags that may be included in the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both include an Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO:29), a calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO:30), a polyglutamic acid tag (EEEEEE; SEQ ID NO:31), an E-tag (GAPVPYPDPLEPR; SEQ ID NO:32), a FLAG-tag (DYKDDDDK; SEQ ID NO:33), an HA-tag, a hemagglutinin-derived peptide (YPYDVPDYA; SEQ ID NO:34), a his-tag (HHHHH (SEQ ID NO:35); HHHHHHH (SEQ ID NO:36); HHHHHHHH (SEQ ID NO:37); HHHHHHHHH (SEQ ID NO:38); HHHHHHHHHH (SEQ ID NO:39); or HHHHHHHHHHH (SEQ ID NO:40)), a myc tag (Glycine, G ... -tag (EQKLISEEDL; SEQ ID NO: 41), NE-tag (TKENPRSNQEESYDDNES; SEQ ID NO: 42), S-tag, (KETAAAKFERQHMDS; SEQ ID NO: 43), SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP; SEQ ID NO: 44), Sof-tag 1 (SLAELLNAGLGGS; SEQ ID NO: 45), Soft-tag 3 (TQDPSRVG; SEQ ID NO: 46), Spot-tag (PDRVRAVSHWSS; SEQ ID NO: 47), Strep-tag (WSHPQFEK; SEQ ID NO: 48), TC-tag (CCPGCC; SEQ ID NO: 49), Ty-tag (EVHTNQDPLD; SEQ ID NO: 50), V5-tag (GKPIPNPLLGLDST; SEQ ID NO: 51), VSV-tag (YTDIEMNRLGK; SEQ ID NO: 52), and Xpress-tag (DLYDDDDK; SEQ ID NO: 53). In some embodiments, the tissue factor protein is a peptide tag.

[0122] A peptide tag, which may be contained in the first chimeric polypeptide of a multi-chain chimeric polypeptide, the second chimeric polypeptide of a multi-chain chimeric polypeptide, or both, can be used in any of a variety of applications related to multi-chain chimeric polypeptides. For example, the peptide tag can be used in purifying the multi-chain chimeric polypeptide. As a non-limiting example, the first chimeric polypeptide of a multi-chain chimeric polypeptide (e.g., a recombinantly expressed first chimeric polypeptide), the second chimeric polypeptide of a multi-chain chimeric polypeptide (e.g., a recombinantly expressed second chimeric polypeptide), or both, can contain a myc tag, and the multi-chain chimeric polypeptide containing the myc-tagged first chimeric polypeptide, the myc-tagged second chimeric polypeptide, or both, can be purified using an antibody that recognizes the myc tag. One non-limiting example of an antibody that recognizes a myc tag is 9E10, which is available from the Developmental Studies Hybridoma Bank, but is not commercially available. As another non-limiting example, a first chimeric polypeptide of a multi-chain chimeric polypeptide (e.g., a recombinantly expressed first chimeric polypeptide), a second chimeric polypeptide of a multi-chain chimeric polypeptide (e.g., a recombinantly expressed second chimeric polypeptide), or both can comprise a histidine tag, and a multi-chain chimeric polypeptide comprising a histidine-tagged first chimeric polypeptide, a histidine-tagged second chimeric polypeptide, or both, can be purified using a nickel or cobalt chelate. Those of skill in the art will be aware of other suitable tags and agents that bind to these tags for use in purifying multi-chain chimeric polypeptides. In some embodiments, the peptide tag is removed from the first chimeric polypeptide and / or the second chimeric polypeptide of the multi-chain chimeric polypeptide after purification. In some embodiments, the peptide tag is not removed from the first chimeric polypeptide and / or the second chimeric polypeptide of the multi-chain chimeric polypeptide after purification.

[0123] The peptide tag, which may be included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of a multi-chain chimeric polypeptide, or both, may be used, for example, in immunoprecipitation of the multi-chain chimeric polypeptide, imaging of the multi-chain chimeric polypeptide (e.g., via Western blotting, ELISA, flow cytometry, and / or immunocytochemistry), and / or solubilization of the multi-chain chimeric polypeptide.

[0124] In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprises a peptide tag that is about 10 to 100 amino acids in length. For example, peptide tags may be about 10 to 100 amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to 100 amino acids in length, about 10 to 95 amino acids in length, about 10 to 90 amino acids in length, about 10 to 85 amino acids in length, about 10 to 80 amino acids in length, about 10 to 75 amino acids in length, The length may be about 10 to 70 amino acids, about 10 to 65 amino acids, about 10 to 60 amino acids, about 10 to 55 amino acids, about 10 to 50 amino acids, about 10 to 45 amino acids, about 10 to 40 amino acids, about 10 to 35 amino acids, about 10 to 30 amino acids, about 10 to 25 amino acids, about 10 to 20 amino acids, about 10 to 15 amino acids, about 20 to 30 amino acids, about 30 to 40 amino acids, about 40 to 50 amino acids, about 50 to 60 amino acids, about 60 to 70 amino acids, about 70 to 80 amino acids, about 80 to 90 amino acids, about 90 to 100 amino acids, about 20 to 90 amino acids, about 30 to 80 amino acids, about 40 to 70 amino acids, about 50 to 60 amino acids, or any range therebetween. In some embodiments, the peptide tag is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

[0125] A peptide tag included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of a multi-chain chimeric polypeptide, or both can be of any suitable length. For example, a peptide tag can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length. In embodiments in which a multi-chain chimeric polypeptide includes two or more peptide tags, the two or more peptide tags can be the same or different lengths. In some embodiments, any of the peptide tags disclosed herein can include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at the N-terminus and / or C-terminus, as long as the function of the peptide tag remains intact. For example, a myc tag having the amino acid sequence EQKLISEEDL (SEQ ID NO: 54) can include one or more additional amino acids (e.g., at the N-terminus and / or C-terminus of the peptide tag) while still retaining the ability to be bound by an antibody.

[0126] Exemplary Multi-Chain Chimeric Polypeptides In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to TGF-β. In some examples of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

[0127] In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are immediately adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

[0128] In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target binding domain are immediately adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target binding domain in the second chimeric polypeptide.

[0129] In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the affinity domain pair can be any of the exemplary affinity domain pairs described herein.

[0130] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain each independently specifically bind to TGF-β. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

[0131] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain are soluble TGF-β receptors (e.g., soluble TGFβRII receptors, e.g., soluble human TGFβRII). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a first sequence of soluble human TGFRβRII and a second sequence of soluble human TGFRβRII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a linker disposed between the first sequence of soluble human TGFRβRII and the second sequence of soluble human TGFRβRII. In some examples of these multi-chain chimeric polypeptides, the linker comprises the sequence GGGGSGGGSGGGGGS (SEQ ID NO: 3).

[0132] In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFRβRII receptor comprises a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to the following: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 2).

[0133] In some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFRβRII receptor comprises a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to the following: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 2).

[0134] In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFRβRII receptor is encoded by a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to the following: ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCA GCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGAC TTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 55).

[0135] In some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFRβRII receptor is encoded by a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to the following: ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTT CCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGAT TTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 56).

[0136] In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor comprises a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to the following: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSIC EKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 4).

[0137] In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical,encoded by an array that is either 100% identical): ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 57).

[0138] In some embodiments, the first chimeric polypeptide can comprise a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: (SEQ ID NO: 6).

[0139]

[0140] In some embodiments, the first chimeric polypeptide can comprise a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: (SEQ ID NO: 7).

[0141]

[0142] In some embodiments, the second chimeric polypeptide can comprise a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to the following: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILED AASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 5).

[0143]

[0144] In some embodiments, the second chimeric polypeptide can comprise a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to the following: MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKL PYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 8).

[0145]

[0146] In some embodiments, the first chimeric polypeptide can comprise a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to the following: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDP KLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 62).

[0147]

[0148] In some embodiments, the first chimeric polypeptide can comprise a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to the following: MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENI TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 64).

[0149]

[0150] In some embodiments, the second chimeric polypeptide can comprise a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: (SEQ ID NO: 66).

[0151]

[0152] In some embodiments, the second chimeric polypeptide can comprise a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: (SEQ ID NO: 68).

[0153]

[0154] Composition / Kit Also provided herein are compositions (e.g., pharmaceutical compositions) comprising at least one of any of the multi-chain chimeric polypeptides, any of the cells, or any of the nucleic acids described herein. In some embodiments, the composition comprises at least one of any of the multi-chain chimeric polypeptides described herein. In some embodiments, the composition comprises any of the immune cells (e.g., any of the immune cells described herein, e.g., any of the immune cells produced using any of the methods described herein).

[0155] In some embodiments, the pharmaceutical composition is formulated for different routes of administration (e.g., intravenous, subcutaneous). In some embodiments, the pharmaceutical composition can include a pharmaceutically acceptable carrier (e.g., phosphate buffered saline).

[0156] Single or multiple administrations of the pharmaceutical composition can be given to a subject in need thereof, depending, for example, on the dosage and frequency as required and tolerated by the subject. The formulation should provide a quantity of the active agent sufficient to effectively treat, prevent, or ameliorate the condition, disease, or symptom.

[0157] Also provided herein are kits comprising any of the multi-chain chimeric polypeptides, compositions, nucleic acids, or cells (e.g., immune cells) described herein. In some aspects, the kits can include instructions for practicing any of the methods described herein. In some embodiments, the kits can include at least one dose of any of the pharmaceutical compositions described herein.

[0158] Nucleic Acids / Vectors Also provided herein are nucleic acids encoding any of the multi-chain chimeric polypeptides described herein. In some embodiments, a first nucleic acid can encode a first chimeric polypeptide, and a second nucleic acid can encode a second chimeric polypeptide. In some embodiments, a single nucleic acid can encode both the first chimeric polypeptide and the second chimeric polypeptide.

[0159] Also provided herein are vectors containing any of the nucleic acids encoding any of the multi-chain chimeric polypeptides described herein. In some embodiments, a first vector can contain a nucleic acid encoding a first chimeric polypeptide, and a second vector can contain a nucleic acid encoding a second chimeric polypeptide. In some embodiments, a single vector can contain a first nucleic acid encoding the first chimeric polypeptide and a second nucleic acid encoding the second chimeric polypeptide.

[0160] Any of the vectors described herein can be an expression vector. For example, the expression vector can include a promoter sequence operably linked to sequences encoding the first and second chimeric polypeptides.

[0161] Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vector (e.g., pSV or pCMV vector), adeno-associated viral (AAV) vector, lentiviral vector, and retroviral vector), as well as any Gateway® vector. A vector can include, for example, sufficient cis-acting elements for expression; other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system. One of skill in the art can select an appropriate vector and mammalian cell for producing any of the multi-chain chimeric polypeptides described herein.

[0162] cell Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the nucleic acids described herein that encode any of the multi-chain chimeric polypeptides described herein (e.g., encoding both the first and second chimeric polypeptides). Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the nucleic acids described herein that encode any of the first chimeric polypeptides described herein. Also provided are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the nucleic acids described herein that encode any of the second chimeric polypeptides described herein.

[0163] Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the vectors described herein encoding any of the multi-chain chimeric polypeptides described herein (e.g., encoding both the first and second chimeric polypeptides). Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the vectors described herein encoding any of the first chimeric polypeptides described herein. Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the vectors described herein encoding any of the second chimeric polypeptides described herein.

[0164] In some embodiments of any of the methods described herein, the cell can be a eukaryotic cell. As used herein, the term "eukaryotic cell" refers to a cell having a separate membrane-bound nucleus. Such cells can include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell such as Saccharomyces cerevisiae. In some aspects, the eukaryotic cell is a higher eukaryote, e.g., a mammalian, avian, plant, or insect cell. Non-limiting examples of mammalia...

Claims

1. 1. A composition for use in treating liver disease, reducing inflammation in the liver, or treating metabolic syndrome in a subject, comprising: (a) Below: (i) a first target binding domain comprising a sequence that is at least 90% identical to SEQ ID NO:4; (ii) a soluble tissue factor domain comprising a sequence that is at least 90% identical to SEQ ID NO:1; and (iii) a first domain of a pair of affinity domains comprising a sequence at least 90% identical to SEQ ID NO: 18; A first chimeric polypeptide comprising: (b) Below: (i) a second domain of the pair of affinity domains comprising a sequence that is at least 90% identical to SEQ ID NO: 16; and (ii) a second target binding domain comprising a sequence that is at least 90% identical to SEQ ID NO:

4. and a second chimeric polypeptide comprising a therapeutically effective amount of a multi-chain chimeric polypeptide comprising Where: the first chimeric polypeptide and the second chimeric polypeptide are associated via binding of a first domain and a second domain of a pair of affinity domains; The composition.

2. 2. The composition of claim 1, wherein the liver disease is selected from the group consisting of fatty liver disease, fatty liver, acute hepatic porphyria, Alagille syndrome, alcohol-related liver disease, alpha-1 antitrypsin deficiency, autoimmune hepatitis, benign liver tumors, cholangiocarcinoma, biliary atresia, Budd-Chiari syndrome, cirrhosis, Crigler-Najjar syndrome, galactosemia, Gilbert syndrome, hemochromatosis, hepatic encephalopathy, hepatitis A, hepatitis B, hepatitis C, hepatorenal syndrome, intrahepatic cholestasis of pregnancy (ICP), lysosomal acid lipase deficiency (LAL-D), hepatic cysts, liver cancer, neonatal jaundice, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis (PFIC), Reye's syndrome, type 1 glycogen storage disease, and Wilson's disease.

3. 2. The composition of claim 1, wherein the metabolic syndrome is selected from the group consisting of coronary heart disease, pulmonary disease, gallbladder disease, dyslipidemia, hypertension, type 2 diabetes, dementia, cancer, gynecological disorders including polycystic ovary syndrome, osteoarthritis, pancreatitis, idiopathic intracranial hypertension, stroke, and cataracts.

4. 1. A composition for use in reducing one or more of the rate of progression of nonalcoholic fatty liver disease (NAFL) to nonalcoholic steatohepatitis (NASH), the progression of NASH to cirrhosis, and the progression of cirrhosis to hepatocellular carcinoma, comprising: (a) Below: (i) a first target binding domain comprising a sequence that is at least 90% identical to SEQ ID NO:4; (ii) a soluble tissue factor domain comprising a sequence that is at least 90% identical to SEQ ID NO:1; and (iii) a first domain of a pair of affinity domains comprising a sequence which is at least 90% identical to SEQ ID NO: 18; A first chimeric polypeptide comprising: (b) Below: (i) a second domain of the pair of affinity domains comprising a sequence that is at least 90% identical to SEQ ID NO: 16; and (ii) a second target binding domain comprising a sequence that is at least 90% identical to SEQ ID NO:

4. and a second chimeric polypeptide comprising a therapeutically effective amount of a multi-chain chimeric polypeptide comprising Where: The first chimeric polypeptide and the second chimeric polypeptide are associated through binding of a first domain and a second domain of a pair of affinity domains; and the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII; The composition.

5. The composition described in claim 4, wherein administration of the composition results in a reduction in the rate of progression of NAFL to NASH.

6. The composition described in claim 4, wherein administration of the composition results in a reduction in the rate of progression of NASH to cirrhosis.

7. The composition described in claim 4, wherein administration of the composition results in a reduction in the rate of progression from cirrhosis to hepatocellular carcinoma.

8. The composition of claim 1 , wherein the subject has previously been identified or diagnosed as having liver disease.

9. 9. The composition of claim 8, wherein the liver disease is selected from the group consisting of fatty liver disease, fatty liver, acute hepatic porphyria, Alagille syndrome, alcohol-related liver disease, alpha-1 antitrypsin deficiency, autoimmune hepatitis, benign liver tumors, cholangiocarcinoma, biliary atresia, Budd-Chiari syndrome, cirrhosis, Crigler-Najjar syndrome, galactosemia, Gilbert syndrome, hemochromatosis, hepatic encephalopathy, hepatitis A, hepatitis B, hepatitis C, hepatorenal syndrome, intrahepatic cholestasis of pregnancy (ICP), lysosomal acid lipase deficiency (LAL-D), hepatic cysts, liver cancer, neonatal jaundice, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis (PFIC), Reye's syndrome, type 1 glycogen storage disease, and Wilson's disease.

10. The composition of claim 1 , wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:

6.

11. The composition of claim 10 , wherein the first chimeric polypeptide comprises the sequence of SEQ ID NO:

6.

12. The composition of claim 11 , wherein the first chimeric polypeptide comprises the sequence of SEQ ID NO:

7.

13. The composition of claim 1 , wherein the second chimeric polypeptide comprises an sequence that is at least 90% identical to SEQ ID NO:

5.

14. The composition of claim 13 , wherein the second chimeric polypeptide comprises the sequence of SEQ ID NO:

5.

15. The composition of claim 14 , wherein the second chimeric polypeptide comprises the sequence of SEQ ID NO:8.

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